Therapeutic aurones

ABSTRACT

Substituted aurones were found to have antitrypanosomal, antifungal and immunomodulatory activity. The invention provides novel aurone compounds, pharmaceutical compositions, and methods encompassing medical and veterinary applications.

This application claims the benefit of U.S. Provisional Application Ser.No. 62/321,079, filed Apr. 11, 2016; 62/321,519, filed. Apr. 12, 2016;62/351,755, filed Jun. 17, 2016; and 62/368,677, filed. Jul. 29, 2016;each of which is incorporated herein by reference in its entirety.

BACKGROUND

Flavonoids are a class of plant and fungus metabolites that possess avariety of biological functions, including pigmentation. Aurones,discovered over seventy years ago, are a naturally, occurring type ofplant flavonoid that lends flowers such as snapdragons and dahlias, abright yellow pigment (Nakayama et al. J Biosci Bioeng, 2002,94:487-491). Perhaps the most notable aurone from plants is aureusidin,extracted from the snapdragon. Some aurones are also known for theirprotection of plants from rot and insects, or for their phytoalexinrole, producing toxins to fight infection (Haudecoeur et al., Curr. Med.Chem. (2012) 19:2861-2875).

Flavonoids typically consist of two phenyl rings (A and B) andheterocyclic ring (C). Aurones differ structurally from flavonoids inthat aurones possess, as the heterocyclic ring (C), a five-membered ring(e.g., a furanone) instead of a six-membered ring (e.g., a pyrone) thatcharacterizes other flavonoids. The benzofuranone ring of an aurone islinked through a carbon-carbon double bond to a phenyl moiety. As arelatively minor plant component, they have only recently attractedresearch attention (Boumendjel, et al., Curr Med Chem, 2003,10:2621-2630; Haudecoeur et al. Curr Med Chem, 2012, 19:2861-2875).

SUMMARY OF THF INVENTION

The present invention provides compounds, compositions, and methods fortreating or preventing infection and disease, for treating or preventingtrypanosomatid infections, fungal infections, and/or inflammatory orimmune diseases or conditions in a vertebrate subject.

In one aspect, the invention provides a compound comprising asubstituted aurone having the stricture of Formula I:

wherein Y is O, N or S;

Z is a substituted aryl group;

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and

a is 0, 1, 2, 3 or 4.

The substituted aurone preferably possesses a biological activity. Inone embodiment, the substituted aurone is effective to treat or preventtrypanosomid infection or disease; i.e., it has anti-trypanosomalactivity. In another embodiment, the substituted aurone is effective totreat or prevent fungal infection or disease; i.e., it has anti-fungalactivity. In yet another embodiment, the substituted aurone is effectiveto treat or prevent an immune disease or condition; i.e., it hasimmunomodulatory activity. A substituted aurone may exhibit one of saidbiological activities, a plurality of said biological activities, or amultiplicity of said biological activities.

In another aspect, the invention provides a method for making asubstituted aurone. In one embodiment, the method includes covalentlylinking a coumaranone and an aldehyde, such as a benzaldehyde or afuraldehyde.

In another aspect, the invention provides a pharmaceutical compositionthat includes as an active agent a substituted aurone. Optionally, thepharmaceutical composition includes a pharmaceutically acceptablecarrier, at least one additional naturally occurring or non-naturallyoccurring active agent (e.g., additional therapeutic agent), or both.For example, in a composition formulated for use in treating a fungalinfection or disease can contain one or more of an azole, a polyene, or5-fluorocytosine.

In another aspect, the invention provides a method involvesadministering to the subject a composition, such as a pharmaceuticalcomposition, containing an effective amount of a compound comprising asubstituted aurone. For example, the composition can be effective totreat or prevent one or more of a trypanosomid infection or disease, afungal infection or disease, or an immune or inflammatory disease orcondition. Any convenient route of administration can be selected. Forexample, administration can be systemic, or localized (e.g., topical).

The subject to whom the active agent is administered can be a human oran animal, such as a companion animal, a domesticated animal, or a wildanimal. Exemplary animals include a dog and a cow (cattle).

In one embodiment, the invention provides a compound comprising asubstituted aurone for use as a prophylactic or therapeutic agent fortreatment or prevention of a trypanosomatid disease or infection, aswell as compositions, such as pharmaceutical compositions, containingthe prophylactic or therapeutic substituted aurone. Also provided is ause of a compound comprising a substituted aurone for preparation of amedicament for the treatment or prevention of trypanosomatid disease orinfection. The invention further provides a method for treating orpreventing a trypanosomatid disease or infection in a subject. In oneembodiment, the method involves administering to the subject aneffective amount of compound comprising a substituted aurone.Representative substituted aurones useful for treating or preventing atrypanosomatid disease or infection include, without limitation,compounds 6620, 6621, 4001, 2014, 9251, 9059, 9087, 9019, 3002, 9024,2023, 9030, 3005, 9028, 7000, 9062, 9065, 3004, 9084, 9067, 2004, 2021,9070, AA8, 2026, 9006, 9057, AA5A, TA2, AA4A, 3001, 9312, AA3A, AA9,2011, 9063, 3012, 6003, 9060, 9078, 9252, 9068, 9061, 2015, 9056, AA11,9086, 7002, 9053, 3009, 9076, 3011, 9058, 8002, 2013, 9029, 6601, 3008,4005, 6617, 2909, 4004, 9064, 9085, 5006, 2904, 9051, 8001, 2911, 2018,6001, 9253, 6000, 9050, 9088, 1009, and 4006. Some substituted aurones,such as compounds 2023, 3002, 6620, 9028, 9030, 9059, 9062, 9065, 9084,9087, and 51, are particularly preferred because they are useful totreat two or more trypanosomid infections. Other substituted auronesuseful in the method of the invention include compounds 2001, 9007,2008, 2906, and 1001, as well as those described in Examples I, II, andIII, and Tables 1, 2, 3A, 3B, and 3C. The trypanosomatid disease orinfection can include, without limitation, human African trypanosomiasis(HAT), animal African trypanosomiasis (AAT), American trypanosomiasis(Chagas Disease) or a leishmaniasis. Exemplary trypanosomatid infectionsinclude a Trypanosoma brucei infection, a Trypanosoma cruzi infection,or a Leishmania infection. Exemplary substituted aurones useful fortreating or preventing a T. brucei infection include, withoutlimitation, compounds 6620, 6621, 4001, 2014. Exemplary substitutedaurones useful for treating or preventing a T. cruzi infection include,without limitation, compounds 9251, 9059, 9087, 9019, 3002, 9024, 2023,9030, 3005, 9028, 7000, 9062, 9065, 3004, 9084. Exemplary substitutedaurones useful for treating or preventing a Leishmania infectioninclude, without limitation, compounds 2023, 9030, 9067, 2004, 2021,9070, AA8, 2026, 9006, 9057, AA5A, 6620, TA2, AA4A, 3001, 9312, AA3A,AA9, 2011, 9063, 3012, 6003, 9060, 9065, 9078, 9252, 9068, 9087, 9062,9061, 2015, 9056, AA11, 9086, 7002, 9053, 9251, 3009, 9076, 9028, 3011,9058, 8002, 9084, 2013, 9029, 6601, 3008, 4005, 6617, 9059, 2909, 4004,9064, 9085, 5006, 2904, 9051, 8001, 3002, 2911, 2018, 6001, 9253, 6000,9050, 9088, 1009, and 4006, as described herein, for example in ExamplesI, II, and III, and Tables 1, 2, 3A, 3B, and 3C.

In another embodiment, the invention provides a compound comprising asubstituted aurone for use as a prophylactic or therapeutic agent fortreatment or prevention of a fungal disease or infection, as well ascompositions, such as pharmaceutical compositions, containing theprophylactic or therapeutic substituted aurone. Also provided is a useof a compound comprising a substituted aurone for preparation of amedicament for the treatment or prevention of fungal disease orinfection. The invention further provides a method for treating orpreventing a fungal disease or infection in a subject. In oneembodiment, the method involves administering to the subject aneffective amount of compound comprising a substituted aurone.Representative substituted aurones useful for treating or preventing afungal disease or infection include, without limitation, compounds 1009and 9051 as described herein, for example in Examples I, IV, and V, andTables 1, 4, and 5.

The fungal infection can include, for example, an infection with Candidaspp., Cryptococcus spp., Saccharomyces spp., or Trichophyton spp. Insome embodiments, the method includes administering an effective amountof a systemic antifungal agent or a topical antifungal agent. In someembodiments, the method includes administering an effective amount of anazole, a polyene, an echinocandin, or 5-fluorocytosine.

In another embodiment, the invention provides an immunomodulatorycompound comprising a substituted aurone for use as a prophylactic ortherapeutic agent for treatment or prevention of immune-relateddiseases, disorders and conditions in a subject, as well as otherconditions accompanied by inflammation, as well as compositions, such aspharmaceutical compositions, containing the prophylactic or therapeuticsubstituted aurone. Also provided is a use of a compound comprising asubstituted aurone for preparation of a medicament for the treatment orprevention of immune-related diseases, disorders and conditions, as wellas other conditions accompanied by inflammation. The invention furtherprovides a method for treating or preventing immune-related diseases,disorders and conditions, as well as other conditions accompanied byinflammation, in a subject. In one embodiment, the method involvesadministering to the subject an effective amount of compound comprisinga substituted aurone that has immunomodulatory activity, termed hereinan “immunomodulatory aurone.” An illustrative immunomodulatory auroneincludes a substituted furyl group and is represented by the structureshown in Formula II:

wherein R₁ is —CH₂OR₄, —CH₂NR₄R₅, —CH₂SR₄, —COR₄, or —CO₂R₄; R₂ and R₃are each independently selected from H, —CH₃, —CH₂OH, —OH or —OCH₃; R₄is H or alkyl; R₅ is H or alkyl; and X and Y are independently selectedfrom O, N and S. In a preferred embodiment, R₂=R₃=H. In anotherpreferred embodiment, R₁ is —CH₂OR₄; more preferably, R₁ is —CH₂OH. Inanother preferred embodiment, at least one of X and Y is O; morepreferably, X=Y=O.

In another illustrative embodiment, the immunomodulatory aurone is(Z)-2-((5-(hydoxymethyl)furan-2-yl)methylene)benzofuran-3(2H)-one; i.e.,Formula (I) where R₁ is —CH₂OH; R₂ and R₃ are both H; and X and Y areboth O; a compound which is referred to herein Aurone 9067 (or, in someinstances, Aurone 1):

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The above summary of the invention is not intended to describe eachdisclosed embodiment or every implementation of the invention. Thedescription that follows more particularly exemplifies illustrativeembodiments. In several places throughout the application, guidance maybe provided through lists of examples, which examples can be used invarious combinations. In each instance, the recited list serves only asa representative group and should not be interpreted as an exclusivelist.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the general structure of an aurone.

FIG. 1B shows the benzofuran-3(2H)-one (3-coumaranone) and benzylidene(styrene) components of an aurone.

FIG. 2 shows an aurone as depicted in FIG. 1A as synthetically derivedfragments: a benzofuranone-derived fragment (BDF) and analdehyde-derived fragment (ADF).

FIG. 3 shows a standard aurone substituent ring numbering scheme.

FIG. 4 shows percent inhibition of C. albicans by 100 μM Aurone 1009,100 μM Aurone 9051, or 100 μM of derivatives of Aurone 1009 or Aurone9051.

FIG. 5 shows the growth curve of Cryptococcus neoformans (Cn) in YPDmedia with and without Aurone 9051 (“Aurone X”).

FIG. 6 shows capsule induction of C. neoformans strain 1199S with andwithout Aurone 9051 (“Aurone X”).

FIG. 7 shows a spotting experiment for C. neoformans (capsule SerotypeD), evaluating the ability of Aurone 9051 to kill cidally or statically.

FIG. 8 shows cytotoxicity of aurone 1 on PMA-differentiated THP-1 andRAW 264.7 macrophages. PMA-differentiated THP-1 or RAW 264.7 cells weretreated with dexamethasone (Dex), Bay11-7082 (Bay), U0126, or theindicated concentrations of aurone 1 in combination with 20 ng/mL LPS.In panel A, the viability of THP-1 cells was measured 4 h post-LPS usingAlamar Blue assay. The same assay was performed for RAW 264.7 cells 4 h(panel B) and 24 h (panel C) post treatment. In all cases, the resultsare presented as the mean±SEM for triplicate measurements from at least3 independent biological repeats. *p<0.05, **p<0.01, ***p<0.001 comparedwith no treatment control group.

FIG. 9 shows that aurone 1 inhibits TNFα, IL-1β, and IL-8 secretion inLPS-stimulated THP-1 cells. PMA-differentiated THP-1 cells werepretreated with dexamethasone (Dex, a synthetic inhibitor of cytokineproduction) as a control or 20, 40, and 80 μM of aurone 1 for 1 h andstimulated with 20 ng/ml of LPS for 4 h. The expression of TNFα (panelA), IL-8 (panel B), and IL-1β (panel C) in supernatants was determinedby ELISA along with calculated fold changes of each cytokine (panel D)Results are presented as the mean±SEM for triplicate measurements of atleast 3 independent experiments. *p<0.05. **p<0.01, ***p<0.001 comparedwith LPS-treated group****p<0.0001 compared with LPS-treated group.

FIG. 10 shows that aurone 1 blocks nuclear translocation of p65 in THP-1cells. PMA-differentiated cells were treated with 50 μM of aurone 1 or10 μM of Bay 11-7082 for 1 h and stimulated with 100 ng/ml LPS for 30min. In panel A, the transcription factor, p65, was stained with rabbitanti-p65 followed by Dylight 488-conjugated secondary antibody (greenfluorescence) and Hoechst 33342 dye (blue fluorescence), sequentially.In panel B, the numeric index of nuclear fluorescence of p65 wascollected using Nuclear Translocation Bioapplication software on theArrayscan VTI reader. *p<0.05, **p<0.01, ***p<0.001 compared withLPS-treated group

FIG. 11 shows that aurone 1 decreases LPS-induced nuclear accumulationof p65 and expression from the 7NE promoter in live murine macrophages.RAW 264.7 cells stably expressing p65-EGFP fusion protein and adestabilized mCherry reporter expressed from the TNFα promoter weretreated with vehicle as a control or 50 μM of aurone 1 for 1 h andstimulated with 20 ng/ml LPS for 6 h. In panel A, time course images ofvehicle and aurone 1-treated RAW 264.7 cells. Fluorescence from p65-EGFPis represented in green and mCherry fluorescence is represented in red.Quantification of p65-EGFP nuclear:cytoplasmic (nuc:cyto) fluorescenceratio for 12 control cells (panel B), 12 aurone-1 pre-treated cells(panel C) is presented together with the population averages (panel D),and the average maximum amplitude of p65-EGFP nuc:cyto fluorescence(panel E). Corresponding measurements of mCherry fluorescence are alsopresented for 12 control cells (panel F), 12 aurone-1 pre-treated cells(panel G), the population averages (panel H), and the average maximummCherry fluorescence (panel I). Data is from a minimum of 44 cellsper-treatment across 3 independent biological repeats. Error ispresented as the SEM. *p<0.05, **p<0.01, ***p<0.001 compared withLPS-treated group.

FIG. 12 shows that aurone 1 inhibits TNFα gene transcription andtransactivation of an NF-κB-dependent promoter. In panel A,PMA-differentiated THP-1 cells were pretreated with 80 μM of aurone 1 orvehicle for 1 h and stimulated with 20 ng/mL of LPS for 4 h. Theexpression of TNFα and B2M snRNA was quantified by qRT-PCR. In panel B,RAW 264.7 cells containing pNT-κB-Luc were treated with 50 μM of aurone1 or vehicle for 1 h and then stimulated with 20 ng/ml LPS for 6 h. Thecells were lysed and the expression of luciferase determined byluminometry. The assay was repeated 3 times as independent experiments.Results are presented as the mean±SEM for triplicate measurements from asingle representative experiment. *p<0.05, **p<0.01, ***p<0.001 comparedwith LPS-treated group LPS-stimulated transcription from anNT-κB-responsive promoter in RAW264.7 cells, RAW 264.7 cells containingpNT-κB-Luc were treated with 50 μM of aurone 1 or vehicle for 1 h andthen stimulated with 20 ng/mL LPS for 6 h. The cells were lysed and theexpression of luciferase determined by luminometry. The assay wasrepeated 3 times as independent experiments. Results are presented asthe mean±SEM for triplicate measurements from a single representativeexperiment. ***p<0.001 as compared to all other conditions.

FIG. 13 shows that aurone 1 inhibits LPS-induced phosphorylation of IKK,p65, and IκBα, and decreases degradation of IκBα. PMA-differentiatedTHP-1 cells and RAW264.7 cells were pretreated with 12.5, 25, and 50 μMof aurone 1 or 10 μM of Bay 11-7082 (Bay) for 1 h and stimulated with 1μg/ml of LPS for 15 min. In panel A, images of blot for phosphorylated(Ser176/180) IKK-αβ and total IKK-β, phosphorylated IκBα (Ser32) andtotal IκBα, and phosphorylated p65 (Ser536) and total p65 in THP-1 cellswere measured by Western blotting followed by densitometry (Panels C, D,and E). Corresponding measurements for pIKK/IKK, pIκBα/actin, andp-p65/p65 in RAW 264.7 cells (panel B) are shown with densitometryresults (Panels F, G and H). Intensity data are represented as themean±SEM for at least three independent experiments. *p<0.05, **p<0.01,***p<0.001 compared with LPS-treated group.

FIG. 14 shows that aurone 1 does not significantly inhibit LPS-inducedphosphorylation of MAPKs. PMA-differentiated THP-1 cells and RAW 264.7cells were pretreated with the indicated concentrations of aurone 1 or10 μM of U0126 for 1 h and stimulated with 1 μg/ml of LPS for 15 min. Inpanel A, image blots of phosphorylated ERK (Thr202/Tyr204) and totalERK, phosphorylated SAPK/JNK (Thr183/Tyr185) and total SAPK/JNK, andphosphorylated p38 (Thr180/Tyr182) and total p38 were measured in THP-1cells by Western blotting followed by densitometry (panels C, D, and E).Corresponding measurements for pERK/ERK, pJNK/JNK, and pp38/β-actin inRAW 264.7 cells (panel B) are shown with densitometry results (panels F,G, and H). Intensity data are represented as the mean±SEM for at leastthree independent experiments. *p<0.05, **p<0.01, ***p<0.001 comparedwith LPS-treated group

FIG. 15 shows that aurone 1 inhibits iNOS expression and NO productionin RAW 264.7 cells. RAW 264.7 cells were pretreated with 12.5, 25, 50,and 100 μM of aurone 1 or 10 μM of Bay 11-7082 for 1 h and stimulatedwith 1 μg/ml of ITS for 24 h. In panel A, actin and iNOS levels weremeasured by Western blotting followed by densitometry. In panel B,nitrite concentration in cell growth medium were analyzed 24 h post LPSby Griess assay as an indirect measurement of NO production. Westernblot intensity data and nitrite concentrations are represented as themean±SEM for at least three independent experiments. *p<0.05, **p<0.01,***p<0.001 compared with LPS-treated group

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure provides compounds, compositions, and methods fortreating or preventing infection and disease in a subject, typically avertebrate subject such as a human, or a wild or domesticated animal, aswell as method for making the compounds.

Aurone derivatives were synthesized to probe the aurone scaffold againstin vitro models for various diseases.

Synthesis of a representative library is reported in Example I. Thelibrary incorporates a wide range of functionality includingbioisosteres and reflects an exploration of different levels oflipophilicity. Structure activity relationships were evaluated to guidefurther optimization of these compounds to treat disease. Compoundshaving anti-trypanosomal, anti-fungal, and/or immunomodulatory activitywere identified.

Accordingly, the present disclosure provides compounds, compositions andmethods relating to aurones, more particularly substituted aurones, andtheir use as a prophylactic or therapeutic agent (i.e., as an activeagent), for example, to prevent or treat parasitic protozoan disease andinfection, to prevent or treat fungal disease or infection, and/orprevent or treat inflammation, autoimmune disease and/or inflammatorydisease. The term “substituted aurone” includes any aurone compoundhaving one or more substituents and/or one or more substitutions of ringatoms. Variants, derivatives, analogs, modifications, and conjugates ofthe substituted aurones described herein, and their use as aprophylactic or therapeutic agents, are also encompassed by theinvention, and the term “substituted aurone” is intended to be inclusiveof active variants, derivatives, analogs, modifications and conjugates.Examples of conjugates include conjugation of aurones to antibodies orantibody fragments, cytokines, chemokines, targeting agents, and thelike. In one embodiment, a substituted aurone is a compound found innature, i.e., a naturally occurring compound. In another, preferredembodiment, a substituted aurone is a compound that is not found innature, i.e., a non-naturally occurring compound. The terms “aurone” and“substituted aurone” are inclusive of aurones wherein in which ringoxygen in the furanone group is replaced with a heteroatom such asnitrogen (to yield an oxindole) or sulfur (to yield a benzothiophenone).Thus, the terms “aurone” and “substituted aurone” include azaaurones andthioaurones. It should be understood that for every embodiment describedherein, the description applies independently and severally to auronestructures that include a ring oxygen, a ring nitrogen, or a ring sulfuras a constituent of the five-membered ring. Substituted aurones can bechemically or enzymatically synthesized. Some of the substituted auronesdescribed herein have novel structures, and the invention should beunderstood to encompass these new compounds as well as methods formaking and using both novel and known substituted aurones, asexemplified throughout the disclosure. Substituted aurones can beadministered alone or in combination with other therapeutics via avariety of routes of administration. Although the invention is describedprimarily with respect substituted aurones, the invention is to beunderstood to encompass related flavonoid structures as would be evidentto one of skill in the art.

Substituted aurones, whether newly discovered or previously known, havebeen surprisingly shown to have biological activity, for example,anti-trypanosomal activity, anti-fungal activity, immunomodulatoryactivity, or a combination thereof.

Substituted Aurones

Aurones are heterocyclic flavonoids characterized by a 15 carbonskeleton containing a coumaranone (benzofuranone) component, or its aza-or thio-counterpart, linked via an exocyclic alkene to an aryl group,for example, another phenyl ring, with the thermodynamically favoredZ-geometry about this alkene (FIG. 1A, reproduced below).

Representative aurones thus contain, as a first component, a coumaranone(benzofuranone) component (typically a 3-coumaranone, also known asbenzofuran-3(2H)-one, or its aza- or thio-counterpart) and, as a second,aryl-containing component, for example a benzylidene (also known as astyrene) component, which contains the exocyclic alkene and an arylgroup (FIG. 1B, reproduced below)

In some aurones, the second, aryl-containing component includes a5-membered ring e.g., furyl) instead of a 6-membered ring (phenyl) asshown above.

In the case of aurones with nitrogen or sulfur substitutions in the fivemembered ring of the first component, it should be understood that thefirst component can be an oxindole or a benzothiophenone.

It should be understood that the first component of the substitutedaurone (i.e., the benzofuranone, oxindole or benzothiophenone) may besubstituted or unsubstituted. However, at least one of the first andsecond components of the substituted aurone is substituted. In someembodiments, an aurone can include a nitrogen or sulfur substitution inthe five membered ring. In such embodiments, the coumaranone componentcan be an oxindole or a benzothiophenone.

In the aurone syntheses described herein, the aryl-containing (e.g.,benzylidene) component of the aurone, designated herein as the secondcomponent of the aurone, is frequently derived from an aldehyde; thus,this second component of the aurone is also referred to herein as an“aldehyde-derived” component or fragment (ADF). The coumaranone(benzofuranone) component (typically a 3-coumaranone, also known asbenzofuran-3(2H)-one, or its aza- or thio-counterpart) of the aurone,designated herein as the first component of the aurone, is analogouslyalso referred to herein as the “benzofuranone-derived” component orfragment (BDF) (FIG. 2, reproduced below).

A substituted aurone is an aurone that contains one or more substituentspositioned at one or more positions on either or both of the first orsecond components of the 15 carbon skeleton, and/or that includes a ringsubstitution.

Roussaki et al. have described the numbering scheme for substituentposition for aurone derivatives (FIG. 3), which is reproduced below toassist in identifying substituent positions (Int. J. Med. Chem. (2012)Article ID 196921).

Compounds of the present invention, structurally based on aurones, werefound to have therapeutic effect. More particularly, these compoundswere found to have anti-trypanosomal, anti-fungal activity,immunomodulatory activity, or combination of said activities.

A representative compound of the invention suitable for use in themethod of the invention is a compound comprising a substituted auronehaving the structure of Formula I:

wherein Y is O, N or S;

Z is a substituted aryl group;

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and

a is 0, 1, 2, 3 or 4.

In some embodiments, Z is selected from the following substituted arylgroups:

wherein X is independently selected from C, O, N, and S;

R1 is selected from substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, nitro, cyano, amine, ester, or combinations thereof; and

b is 0, 1, 2, 3, or 4.

It will be understood that the term “substituted aryl group” as usedherein is inclusive of a heteroaryl group, wherein the heteroatom istreated as a substitution. In embodiments wherein Z is a substitutedheteroaryl group, the heteroaryl group may be further substituted (i.e.,b>0), but need not be further substituted (i.e., b=0), to include one ormore ring substituents.

Representative substituted aurones having formula I in which Y is Oinclude the following:

wherein Z is selected from:

X is independently selected from C, O, N, and S;

R1 is selected from substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, nitro, cyano, amine, ester, or combinations thereof;

b is 0, 1, 2, 3, or 4;

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers; or combinations thereof;and

a is 0, 1, 2, 3 or 4.

Representative substituted aurones having Formula I in which Y is N(azaaurones) include the following:

wherein Z is selected from:

X is independently selected from C, O, N, and S;

R1 is selected from substituted or unsubstituted alkyl, substituted orunsubstituted a substituted or unsubstituted alkoxy, hydroxyl, halogen,nitro, cyano, amine, ester, or combinations thereof;

b is 0, 1, 2, 3, or 4;

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and

a is 0, 1, 2, 3 or 4.

In another embodiment, a substituted aurone suitable for use in themethod of the invention has the structure of Formula I:

wherein Y is O, N or S;

Z is selected from:

R1 is selected from substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, nitro, cyano, amine, ester, or combinations thereof;

b is 0, 1, 2, 3, or 4;

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and

a is 0, 1, 2, 3 or 4.

In a preferred embodiment, the substituted aurone suitable for use inthe method of the invention has the structure of Formula I:

wherein Z is

R1 is selected from halogen, cyano, halogen substituted alkyl, orcombinations thereof;

b is 1 or 2;

R2 is halogen; and

a is 0 or 1.

In another preferred embodiment, the substituted aurone suitable for usein the method of the invention has the structure of Formula I:

wherein Z is

-   -   R1 is selected from halogen, cyano, or combinations thereof;    -   b is 1 or 2;    -   R2 is halogen; and    -   a is 0 or 1.

Additional newly discovered compounds, suitable for use in the method ofthe invention, include the compound of Formula I:

wherein Z selected from

where R1 is selected from iodine (I) or trifluoromethyl (CF₃), and b is1, 2 or 3;

where R1 is selected from alkyl, hydroxyl substituted alkyl, orcombinations thereof and b is 1 or 2;

where R1 is selected from halogen, or combinations thereof and b is 1 or2; and

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkoxy hydroxyl, halogen, or combinations thereof; and

a is 0, 1, 2, 3 or 4.

A preferred embodiment of the compound of Formula I is as follows:

wherein Z is selected from:

where R1 is selected from iodine (I) or trifluoromethyl (CF₃), and b is1, 2 or 3;

where R1 is selected from a C₁ to C₄ alkyl, hydroxyl substituted C₁ toC₄ alkyl, or combinations thereof and b is 1;

where R1 is bromine and b is 1 or 2; and

R2 is substituted or unsubstituted alkyl; and

a is 0 or 1.

Exemplary substituted aurones for use in the method of the inventioninclude, without limitation, substituted aurones listed in Tables 1, 2,3A, 3B, 3C, 4A, 4B, 5, 8, 9, and 10.

As used herein, “alkyl” refers to an unsubstituted or substitutedsaturated hydrocarbon chain radical having from 1 to about 15 carbonatoms; from 1 to about 10 carbon atoms; from 1 to about 6 carbon atoms;or from 1 to about 4 carbon atoms. Non-limiting examples of alkyl groupsinclude, for example, methyl, ethyl, propyl, iso-propyl, and butyl.

As used herein, “alkenyl” refers to an unsubstituted or substitutedhydrocarbon chain radical having at least one carbon-carbon double bondand having from about 2 to about 15 carbon atoms; from 2 to about 10carbon atoms; or from 2 to about 8 carbon atoms. Non-limiting examplesof alkenyls include, for example, vinyl, allyl, and butenyl.

As used herein, “alkynyl” “refers to an unsubstituted or substitutedhydrocarbon chain radical having at least one carbon-carbon triple bond,and having from about 2 up to about 15 carbon atoms; from 2 to about 10carbon atoms; or from about 2 to about 8 carbon atoms. Non-limitingexamples of alkynyls include, for example ethynyl, propynyl, propargyland butynyl.

As used herein, “aryl.” refers to an aromatic, carbocyclic orheterocyclic ring radical. Non-limiting examples of aryls include, forexample, phenyl, tolyl, xylyl, cumenyl, naphtyl, biphenyl, thienyl,furyl, pyrrolyl, pyridinyl, pyrazinyl, thiazolyl, pyrimidinyl,quinolinyl, tetrazolyl, benzothiazolyl, benzofuryl, indolyl, and thelike. Aryls may be substituted or unsubstituted.

As used herein “alkoxy” refers to an alkyl, alkenyl, or alkynyl group,as defined herein, attached to an oxygen radical. The term “alkoxy” alsoincludes alkyl ether groups, where the term ‘alkyl’ is defined above,and ‘ether’ means two alkyl groups with an oxygen atom between them.Non-limiting examples of alkoxy groups include methoxy, ethoxy,n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, methoxymethane (alsoreferred to as ‘dimethyl ether’), and methoxyethane (also referred to as‘ethyl methyl ether’).

As used herein, “hydroxyl group” or “hydroxyl” refers to a substituentgroup of formula. —OH.

As used herein, “halogen” or “halide” refers to fluoride, chloride,bromide or iodide. The terms “fluoro”, “chloro”, “bromo”, and “iodo” mayalso be used when referring to halogenated substituents, for example,“trifluoromethyl.”

As used herein, “amine group” has the general formula —NRR, here each Ris independently hydrogen, or a hydrocarbon.

As used herein, “cyano group” or “cyano” refers to a —CN group.

The term “azido group” or “azido”, refers to an —N₃ group.

As used herein, “ether group” or “ether” refers to radicals of thegeneral formula —R′—O—R″, where R and R″ are independently substitutedor unsubstituted hydrocarbyl.

As used herein “nitro group” or “nitro” refers to —NO₂.

As used herein “ester group” or “ester” refers to a substituent of thegeneral formula —C—O—O—R¹ where R¹ may be either aliphatic or aromatic.

The term substituted refers to the moiety (e.g., alkyl, alkenyl,cycloalkyl, aryl, etc.) bearing one or more substituents. Non-limitingexamples of substituents can include alkyl, alkenyl, alkynyl, hydroxyl,alkoxy, heterocyclic, aryl, heteroaryl, aryloxy, halogen, haloalkyl,cyano, nitro, amino, lower alkylamino, lower dialkylamino, amido, azido,acyl (—C(O)R₆), carboxyl (—C(O)OH), ester (—C(O)OR₆), carbamate (—OC(O)N(R₆)₂), wherein R₆ is H or lower alkyl, lower alkenyl, lower alkynyl,aryl, heteroaryl, heterocycle, and the like. In the case of an auronering structure, the term “substituted” can include the substitution of aheteroatom into the aurone ring structure.

Compounds described herein, which may be suitable for use in one or moremethods described herein, include both newly discovered compounds aswell as compounds that may be known to the art, but not heretofore knownto possess the activity or activities described herein. Exemplarysubstituted aurones that are believed to be novel, also shown by thenumerical designations in Table 1, may include, but are not limited to,a compound selected from:

In some embodiments of the substituted aurone, preferred substituents onthe first (BDF) component include methyl and halo. In some embodimentsof the substituted aurone, preferred substituents on the second (NEW)component include cyano and halo. In a preferred embodiment of asubstituted aurone, one or more substituents does not include an oxygenatom. In a particularly preferred embodiment, none of the substituent(s)contains an oxygen atom.

It should be understood that in instances where the substituted auronesare exemplified using a benzofuranone as the first (BDF) component,analogous compounds containing, as the first component, anitrogen-containing oxindole, or a sulfur-containing benzothiophenone,in place of the benzofuranone, are likewise encompassed by theinvention.

Synthesis of Substituted Aurones

While the ring-closure of chalcones is one route to aurones, it is notalways reliable and often employs highly toxic reagents and/or solvents.A more general approach has been the condensation of a coumaranone withan aldehyde:

Many different reaction conditions can be employed for this reaction,the most typical being highly basic (potassium hydroxide in methanol orsodium methoxide in methanol) (Varma et al., Tetrahedron Letters. 1992,17, 5937-5940). At the same time, these conditions can prove to beincompatible with certain functional groups. Even the conditionsreported by Varma using alumina can be difficult to scale up and canoffer poorly reproducible results (Lee et al., Eur. J. Med. Chem. 452957-2971). The neutral conditions reported recently by Handy andHawkins using the deep eutectic solvent comprised of a 1:2 molar ratioof choline chloride and urea have proven to be far more general and haveopened new opportunities for the study of aurone derivatives (Hawkins etal., Tetrahedron 2013, 69, 9200-9204).

Several non-limiting methods for synthesizing substituted aurones areexemplified as Methods 1 through 7 in Example I.

Representative Aurones Exhibiting Anti-Trypanosomal Activity

Members of the library of aurones were evaluated for anti-trypanosomalactivity (see, e.g., Examples II and III). It has been surprisinglyfound that substituted aurones have antitrypanosomal activity and arethus well-suited for medical and veterinary applications, boththerapeutic and prophylactic. Preferred substituted aurones of theinvention are those that exhibit high parasite inhibition (e.g.,antittypanosomal activity) and, optionally, low toxicity. An exemplarysubstituted aurone of the invention can have an IC₅₀ value of <100 μM,<50 μM, <40 μM, <30 μM, <20 μM, <10 μM, <5 μM, <3 μM or <1 μM against aparasitic pathogen, for example against a trypanosomal pathogen such asT. brucei, T. cruzi, or Leishmania spp., such as Leishmania amazonensis(see, e.g., inhibition assays described in Example II and Tables 1 and2). Optionally, a substituted aurone of the invention exhibits aselectivity multiple (parasite inhibition vs mammalian cell toxicity, asdescribed in more detail below) of greater than 4, preferably greaterthan 10. Alternatively or additionally, the mean percent inhibition ofthe substituted aurone at a dosing level of 50 μM is >50% and,optionally, the toxicity <10%. Exemplary assays for evaluatingantitrypanosomal activity (e.g., using T. brucei, T. cruzi, or L.amazonensis) and toxicity (e.g., evaluating selectivity using themammalian cell toxicity model L6) are described in Example II.Substituted aurones can be conveniently compared to the parent auronecompound, compound 6615, to assess improved antitrypanosomal activityand/or selectivity or other measure of toxicity.

Exemplary compounds having antitrypanosomal activity are identifiedthroughout the disclosure, including for example in Tables 1, 2, 3A, 3B,and 3C, and include, without limitation, compounds 6620, 6621, 4001,2014, 9251, 9059, 9087, 9019, 3002, 9024, 2023, 9030, 3005, 9028, 7000,9062, 9065, 3004, 9084, 9067, 2004, 2021, 9070, AA8, 2026, 9006, 9057,AA5A, TA2, AA4A, 3001, 9312, AA3A, AA9, 2011, 9063, 3012, 6003, 9060,9078, 9252, 9068, 9061, 2015, 9056, AA11, 9086, 7002, 9053, 3009, 9076,3011, 9058, 8002, 2013, 9029, 6601, 3008, 4005, 6617, 2909, 4004, 9064,9085, 5006, 2904, 9051, 8001, 2911, 2018, 6001, 9253, 6000, 9050, 9088,1009, and 4006. Some substituted aurones, such as compounds 2023, 3002,6620, 9028, 9030, 9059, 9062, 9065, 9084, 9087, and 9251, areparticularly preferred because they are useful to treat two or moretrypanosomid infections. Other substituted aurones useful in the methodof the invention include compounds 2001, 9007, 2008, 2906, and 1001, aswell as those described in Examples I, II, and III, and Tables 1, 2, 3A,3B, and 3C. Exemplary substituted aurones useful for treating orpreventing a T. brucei infection include, without limitation, compounds6620, 6621, 4001, 2014. Exemplary substituted aurones useful fortreating or preventing a T. cruzi infection include, without limitation,compounds 9251, 9059, 9087, 9019, 3002, 9024, 2023, 9030, 3005, 9028,7000, 9062, 9065, 3004, 9084. Exemplary substituted aurones useful fortreating or preventing a Leishmania infection include, withoutlimitation, compounds 2023, 9030, 9067, 2004, 2021, 9070, AA8, 2026,9006, 9057, AA5A, 6620, TA2, AA4A, 3001, 9312, AA3A, AA9, 2011, 9063,3012, 6003, 9060, 9065, 9078, 9252, 9068, 9087, 9062, 9061, 2015, 9056,AA11, 9086, 7002, 9053, 9251, 3009, 9076, 9028, 3011, 9058, 8002, 9084,2013, 9029, 6601, 3008, 4005, 6617, 9059, 2909, 4004, 9064, 9085, 5006,2904, 9051, 8001, 3002, 2911, 2018, 6001, 9253, 6000, 9050, 9088, 1009,and 4006.

Representative Aurones Exhibiting Anti-Fungal Activity

Members of the library of aurones were evaluated for anti-fungalactivity (see, e.g., Example IV and V). Surprisingly, Aurone 1009 andAurone 9051 were found to have significant antifungal activity against anumber of the most problematic Candida pathogens. The IC50 values forAurone 1009 and Aurone 9051 ranged from 10 μM to 18 μM against C.albicans, C. glabrata, and C. tropicalis. In addition. Aurone 1009 andAurone 9051 were found to have significant antifungal activity againstCryptococcus neoformans with MIC values of 30 μM for Aurone 1009 and 110μM for Aurone 9051. As shown in Example IV and Table 4, Aurone 1009 andAurone 9051 demonstrate significantly greater C. albicans inhibitioncompared to other aurones even aurones having similar structures.

Exemplary compounds with anti-fungal activity thus include Aurone 1009and Aurone 9051. The structures of Aurone 1009 and Aurone 9051 are asfollows:

Because Aurone 1009 and Aurone 9051 are easily synthesized and exhibitsignificant antifungal activity and low toxicity against humans cells,these aurones are particularly promising as therapeutic agents for thetreatment of fungal infections.

Representative Aurones Exhibiting Immunomodulatory Activity

Members of the library of aurones were evaluated for immunomodulatoryactivity (see, e.g., Example VI). Aurones 9067 (referred to in ExampleVI as “Aurone 1”), aurone 9251 (referred to in Example VI as “Aurone 2”)and aurone 2023 (referred to in Example VI as “Aurone 3”) described inExample VI, as well as the aurone of Formula II, have a backbonestructure that is somewhat different from a typical aurone; they arecharacterized by a 13 carbon skeleton rather than a 15 carbon skeletonbecause they contain, as the B ring, a 5-membered heterocyclic furylgroup instead of a 6-membered aryl group (e.g., phenyl).

An immunomodulatory aurone preferably contains, as the B ring in thesecond component, a substituted furyl group. An exemplaryimmunomodulatory aurone is shown in Formula II:

wherein R₁ is —CH₂OR₄, —CH₂NR₄R₅, —CH₂SR₄, or —CO₂R₄; R₂ and R₃ are eachindependently selected from H, —CH₃, —CH₂OH, —OH or —OCH₃; R₄ is H oralkyl; R₅ is H or alkyl; and X and Y are independently selected from O,N and S. In a preferred embodiment, R₂=R₃=H. In another preferredembodiment, R₁ is —CH₂OR₄; more preferably, R₁ is —CH₂OH. In anotherpreferred embodiment, at least one of X and Y is O; more preferably,X=Y=O. As used herein, “alkyl” refers to an unsubstituted or substitutedsaturated hydrocarbon chain radical having from 1 to about 15 carbonatoms; from 1 to about 10 carbon atoms; from 1 to about 6 carbon atoms;or from 1 to about 4 carbon atoms. Non-limiting examples of alkyl groupsinclude, for example, methyl, ethyl, propyl, iso-propyl, butyl,iso-butyl, sec-butyl and tort-butyl.

A particularly preferred compound is(Z)-2-((5-(hydoxymethyl)furan-2-yl)methylene)benzofuran-3(2H)-one; i.e.,Formula (I) where R₁ is —CH₂OH; R₂ and R₃ are both H; and X and Y areboth O. This compound is referred to herein as Aurone 9067:

Aurone 9067, in which the benzofuranone component is unsubstituted, andwhich contains a single hydroxymethyl substituent at position C-5′ onthe furan-2-yl ring (as R₁ of Formula II), is a novel synthetic auronethat was discovered to have significant immunomodulator activity for thetreatment of immune based-conditions or diseases (see, e.g., ExampleVI); and is thus very promising as a therapeutic agent. Aurone 9067 hasvery low cytotoxicity, along with potent immunomodulatory activity inthe low μM range. Exemplary synthetic protocols for Aurone 9067 as wellas other aurones are described in Example VI. Typically, theimmunomodulatory aurone is synthesized by covalently linking acoumaranone and an aldehyde, wherein the aldehyde is chosen to yield theaurone of interest. For example, the aldehyde can be a furaldehyde, suchas a hydroxymethylfuraldehyde.

Aurone 9067 contains a single substituent (hydroxymethyl) at the 5′position of the furyl ring. Compounds having one or two additionalsubstituents at the 4′ position and/or the 3′ position of the furyl ringmay also exhibit immunomodulatory activity. Additional substituent(s) atthe 3′ and/or 4′ ring positions can be independently selected frommethyl, hydroxymethyl, hydroxyl and methoxy. For example, a compoundhaving —CH₂OH at position C-5′ as well as one or both of positions C-4′and C-3′ of the furyl ring may exhibit immunomodulatory activity. Insome embodiments, the aurone can include a nitrogen or sulfursubstitution for the oxygen atom in the five membered heterocyclic ringthat is included in the first, coumaranone component (C ring). In suchembodiments, which encompass the aza- or thio-counterpart of the aurone,the first component can be an oxindole or a benzothiophenone.Alternatively or additionally, in some embodiments, the aurone caninclude a nitrogen or sulfur substitution for the oxygen atom in the5-membered heterocyclic ring that forms part of the second,furyl-containing component (B ring).

Pharmaceutical Compositions

The present disclosure provides a pharmaceutical composition thatincludes, as an active agent, a substituted aurone, and apharmaceutically acceptable carrier. In exemplary embodiments, thesubstituted aurone includes an aurone according to Formula I or Formulaand may include one or more of aurone compounds 6620, 6621, 4001, 2014,9251, 9059, 9087, 9019, 3002, 9024, 2023, 9030, 3005, 9028, 7000, 9062,9065, 3004, 9084, 2004, 2021, 9070, AA8, 2026, 9006, 9057, AA5A, TA2,AA4A, 3001, 9312, AA3A, AA9, 2011, 9063, 3012, 6003, 9060, 9078, 9252,9068, 9061, 2015, 9056, AA11, 9086, 7002, 9053, 3009, 9076, 3011, 9058,8002, 2013, 9029, 6601, 3008, 4005, 6617, 2909, 4004, 9064, 9085, 5006,2904, 9051, 8001, 2911, 2018, 6001, 9253, 6000, 9050, 9088, 1009, and4006, 1009, 9051, 9067, 2001, 9007, 2008, 2906, and 1001, as well asthose described in Tables 1, 2, 3A, 3B, 3C, 4A, 4B, 5, 8, 9, and 10.

The active agent is formulated in a pharmaceutical composition and then,in accordance with the method of the invention, administered to avertebrate, particularly a mammal, such as a human, a domestic orcompanion animal, a zoo animal, a research animal, or a domesticatedanimal, such as a farm animal, in a variety of forms adapted to thechosen route of administration. The formulations include those suitablefor oral, rectal, vaginal, topical, nasal, ophthalmic or parenteral(including subcutaneous, intramuscular, intraperitoneal, andintravenous) administration.

The pharmaceutically acceptable carrier can include, for example, anexcipient, a diluent, a solvent, an accessory ingredient, a stabilizer,a protein carrier, or a biological compound. Non-limiting examples of aprotein carrier includes keyhole limpet hemocyanin (KLH), bovine serumalbumin (BSA), ovalbumin, or the like. Non-limiting examples of abiological compound which can serve as a carrier include aglycosaminoglycan, a proteoglycan, and albumin. The carrier can be asynthetic compound, such as dimethyl sulfoxide or a synthetic polymer,such as a polyalkyleneglycol. Ovalbumin, human serum albumin, otherproteins, polyethylene glycol, or the like can be employed as thecarrier. In a preferred embodiment, the pharmaceutically acceptablecarrier includes at least one compound that is not naturally occurringor a product of nature.

In some embodiments, the substituted aurone is formulated in combinationwith one or more additional (i.e., “second”) active agents. For example,a substituted aurone with anti-trypanosomal activity can be formulatedin combination with another antiprotozoan and/or antiparasitic compound,or with an immunomodulatory agent, including an immunomodulatory aurone.As another example, a substituted aurone with anti-fungal activity canbe formulated in combination with azole, a polyene, 5-fluorocytosine,and/or an echinocandin, or with an immunomodulatory agent, including animmunomodulatory aurone. As another example, a substituted aurone of theinvention having one or more of anti-trypanosomal, anti-fungal orimmunomodulatory activity can be optionally combined with otherprophylactic, therapeutic or palliative agents such as ananti-inflammatory agent, a cytokine, a chemokine, a therapeuticantibody, an immunogen, an antigen, an adjuvant, or an antioxidant, animmunomodulatory compound, an analgesic, a non-steroidalanti-inflammatory drug, a biologic compound, an antineoplastic agent,anticancer agent, antiangiogenic agent, a chemopreventive agent, or achemotherapeutic agent.

Examples of immunomodulatory compounds and biologics that can be used incombination therapy with a substitute aurone, such as animmunomodulatory aurone as set forth herein (whether administered in asingle pharmaceutical composition with the immunomodulatory aurone, orseparately) are throughout the disclosure, including Example VI. Moregenerally, any known therapeutic or prophylactic agent can be includedas additional active agent. The action of the additional active agent inthe combination therapy can be cumulative to the substituted aurone orit can be complementary, for example to manage side effects or otheraspects of the patient's medical condition, such as pain, swelling, andthe like.

An exemplary multicomponent composition is a vaccine. A vaccine containsat least one immunogenic or antigenic component, and a pharmaceuticallyacceptable carrier. Optionally, a vaccine includes one or moreadjuvants. An immunomodulatory aurone can be included in a vaccinecomposition to ameliorate, reduce, or eliminate a reactogenicinflammatory response in the subject to whom the vaccine isadministered. Inclusion of an immunomodulatory aurone in vaccineformulations may reduce reactogenicity, particularly in live virusvaccines. See Athearn et al., PLoS One. 2012; 7(10):e46516. doi:10.1371/journal.pone.0046516. Epub 2012 Oct. 8; Lewis et al., J ImmunolRes. 2015; 2015:909406. Epub 2015 Aug. 25). More generally, animmunomodulatory aurone can be co-administered with therapeutic agentsthat might otherwise trigger inflammation, particularly in sensitive,ill or vulnerable individuals, such as the very young or very old, inorder to reduce the extent of the inflammatory response.

In one embodiment, the combination therapy includes at least onecompound that is not naturally occurring or a product of nature. In aparticularly preferred embodiment, the pharmaceutical compositionincludes at least one non-naturally occurring therapeutic orprophylactic agent.

The formulations can be conveniently presented in unit dosage form andcan be prepared by any of the methods well-known in the art of pharmacy.AR methods include the step of bringing the active agent intoassociation with a pharmaceutical carrier. In general, the formulationsare prepared by uniformly and intimately bringing the active compoundinto association with a liquid carrier, a finely divided solid carrier,or both, and then, if necessary, shaping the product into the desiredformulations.

Formulations of the present invention suitable for oral administrationcan be presented as discrete units such as tablets, troches, capsules,lozenges, wafers, or cachets, each containing a predetermined amount ofthe active agent as a powder or granules, as liposomes, or as a solutionor suspension in an aqueous liquor or non-aqueous liquid such as asyrup, an elixir, an emulsion, or a draught. The tablets, troches,pills, capsules, and the like can also contain one or more of thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; an excipient such as dicalcium phosphate; a disintegratingagent such as corn starch, potato starch, alginic acid, and the like; alubricant such as magnesium stearate; a sweetening agent such assucrose, fructose, lactose, or aspartame; and a natural or artificialflavoring agent. When the unit dosage form is a capsule, it can furthercontain a liquid carrier, such as a vegetable oil or a polyethyleneglycol. Various other materials can be present as coatings or tootherwise modify the physical form of the solid unit dosage form. Forinstance, tablets, pills, or capsules can be coated with gelatin, wax,shellac, sugar, and the like. A syrup or elixir can contain one or moreof a sweetening agent, a preservative such as methyl- or propylparaben,an agent to retard crystallization of the sugar, an agent to increasethe solubility of any other ingredient, such as a polyhydric alcohol,for example glycerol or sorbitol, a dye, and flavoring agent. Thematerial used in preparing any unit dosage form is substantiallynontoxic in the amounts employed. The active agent can be incorporatedinto preparations and devices in formulations that may, or may not, bedesigned for sustained release or controlled release.

Formulations suitable for parenteral administration conveniently includea sterile aqueous preparation of the active agent, or dispersions ofsterile powders of the active agent, which are preferably isotonic withthe blood of the recipient. Parenteral administration a substitutedaurone (e. g., through an I. V. drip) is one form of administration.Isotonic agents that can be included in the liquid preparation includesugars, buffers, and sodium chloride. Solutions of the active agent canbe prepared in water, optionally mixed with a nontoxic surfactant.Dispersions of the active agent can be prepared in water, ethanol, apolyol (such as glycerol, propylene glycol, liquid polyethylene glycols,and the like), vegetable oils, glycerol esters, and mixtures thereof.The ultimate dosage form is sterile, fluid, and stable under theconditions of manufacture and storage. The necessary fluidity can beachieved, for example, by using liposomes, by employing the appropriateparticle size in the case of dispersions, or by using surfactants.Sterilization of a liquid preparation can be achieved by any convenientmethod that preserves the bioactivity of the active agent, preferably byfilter sterilization. Preferred methods for preparing powders includevacuum drying and freeze drying of the sterile injectable solutions.Subsequent microbial contamination can be prevented using variousantimicrobial agents, for example, antibacterial, antiviral andantifungal agents including parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. Absorption of the active agents over aprolonged period can be achieved by including agents for delaying, forexample, aluminum monostearate and gelatin.

Nasal spray formulations include purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes. Formulations for rectal orvaginal administration can be presented as a suppository with a suitablecarrier such as cocoa butter, or hydrogenated fats or hydrogenated fattycarboxylic acids. Ophthalmic formulations are prepared by a similarmethod to the nasal spray, except that the pH and isotonic factors arepreferably adjusted to match that of the eye. Topical formulationsinclude the active agent dissolved or suspended in one or more mediasuch as mineral oil, petroleum, polyhydroxy alcohols, or other basesused for topical pharmaceutical formulations. Topical formulations canbe provided in the form of a bandage, wherein the formulation isincorporated into a gauze or other structure and brought into contactwith the skin.

Administration of Substituted Aurones

A substituted aurone, as the active agent, can be administered to asubject alone or in a pharmaceutical composition that includes theactive agent and a pharmaceutically acceptable carrier. The term“administered” encompasses administration of a prophylactically and/ortherapeutically effective dose or amount of the active agent to asubject. The active agent is administered to a vertebrate, particularlya mammal, such as a human, a domestic or companion animal, a zoo animal,a research animal, or a domesticated animal, such as a farm animal, inan amount effective to produce the desired effect. The term “effectivedose” or “effective amount” refers to a dose or amount that produces theeffects for which it is administered, especially an intended effect suchas an anti-trypanosomal effect, and anti-fungal effect, orimmunomodulatory or anti-inflammatory effect.

A substituted aurone can be introduced into the subject systemically orlocally, for example at the site of infection or inflammation. Theactive agent is administered to the subject in an amount effective toproduce the desired effect. A substituted aurone can be administered ina variety of routes, including orally, parenterally, intraperitoneally,intravenously, intraarterially, transdermally, sublingually,intramuscularly, rectally, transbuccally, intranasally, liposomally, viainhalation, vaginally, intraoccularly, via local delivery by catheter orstent, subcutaneously, intraadiposally, intraarticularly, intrathecally,or in a slow release dosage form. Local administration can includetopical administration, administration by injection, or perfusion orbathing of an organ or tissue, for example.

The formulations can be administered as a single dose or in multipledoses. Useful dosages of the active agents can be determined bycomparing their in vitro activity and the in vivo activity in animalmodels. Methods for extrapolation of effective dosages in mice, andother animals, to humans are known in the art.

Dosage levels of the active agent in the pharmaceutical compositions ofthis invention can be varied so as to obtain an amount of the activeagent which is effective to achieve the desired therapeutic response fora particular subject, composition, and mode of administration, withoutbeing toxic to the subject. The selected dosage level will depend upon avariety of factors including the activity of the particular compound ofthe present invention employed, or the ester, salt or amide thereof, theroute of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the substituted aurone, the age, sex, weight, condition, generalhealth and prior medical history of the subject being treated, and likefactors well known in the medical arts.

In the case of substituted aurones with anti-trypanosomal activity,dosages and dosing regimens that are suitable for other prophylactic andtherapeutic anti-protozoan agents are likewise suitable for therapeuticor prophylactic administration of a substituted aurone. For example,dosages or dosing regimens in use for other plant-derived compounds,such as the antimalarial botanical artemisinin, may serve as guidepostsfor developing suitable animal and human dosages and dosing regimens.Examples of other antiparasitic therapies which can form the basis fordetermining dosages and dosing regimens for a substituted aurone can befound in Kappagoda et al., “Antiparasitic Therapy,” Mayo Clin. Proc.2011, 86(6)561-583.

In the case of substituted aurones with anti-fungal activity, dosagesand dosing regimens that are suitable for other prophylactic andtherapeutic anti-fungal agents are likewise suitable for therapeutic orprophylactic administration of an aurone. For example, dosages or dosingregimens in use for anti-fungal compounds, including, for example,flucytosine, may serve as guideposts for developing suitable animal andhuman dosages and dosing regimens.

In the case of substituted aurones having immunomodulatory activity,dosages and dosing regimens that are suitable for other flavonoids orsimilar compounds are suitable for therapeutic or prophylacticadministration of the immunomodulatory aurone. The dosage in bothnutraceutical or pharmaceutical use typically is such that the amount ofthe immunomodulatory aurone administered to a subject is such that it iseffective reduce inflammation or have other beneficial effect.

In exemplary administrations, a substituted aurone can be administeredto a subject orally, in an amount of between 5 mg and 100 mg, or between10 mg and 100 mg, at least once per day, as a medication, nutritionalsupplement, or food additive. As another example, a substituted auronecan be administered to a subject in dosages ranging from 0.01 mg/kg to10 mg/kg body weight, or 0.1 mg/kg to 20 mg/kg body weight, or higher;or in a form sufficient to provide a daily dosage of 0.01 mg/kg to about20 mg per/kg body weight, or 0.03 mg/kg to about 10 mg/kg body weight ofthe subject, to which it is to be administered. As a further example, asubstituted aurone can be administered to a subject intravenously orintramuscularly in an amount between 5 mg and 100 mg at least once perday. As yet another example, a substituted aurone can be administered ina daily dose of about 0.2 g to 1000 g; for example, 0.5 g to 5 g can beadministered to a person with a weight of 70 kg per day in one or more,e.g. 1 to 3, dosages, and the amount administered can be adjusted forthe weight of the subject. See PCT Publication WO 2010110646 A1; seealso U.S. Pat. Publ. 20080262081 for nutraceutical compositions, dosinginformation, and methods relating to resveratrol that can be used fordosing guidance.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician could start doses ofthe substituted aurone of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

Methods to Treat or Prevent Parasitic Disease or Infection

Substituted aurones can be used to treat or prevent parasiticinfections, particularly protozoan infections caused by trypanosomes.The terms “trypanosome,” “trypanosomatid” and “trypanosomal” are used toindicate or describe organisms, or human or animal diseases caused byprotozoa in the family Trypanosomatidae which includes the generaTrypanosoma and Leishmania. Exemplary trypanosomatid infections include,but are not limited to, human African trypanosomiasis (HAT), animalAfrican trypanosomiasis (AAT), American trypanosomiasis (ChagasDisease), and leishmaniasis. The invention provides a therapeutic methodof treating a subject suffering from infection with a parasiticprotozoan by administering a substituted aurone to the subject.Therapeutic treatment is initiated after diagnosis or the development ofsymptoms of infection with a parasitic protozoan.

A substituted aurone can also be administered prophylactically, toprevent or delay the development of infection with a parasiticprotozoan. Treatment that is prophylactic, for instance, can beinitiated before a subject manifests symptoms of infection with aparasitic protozoan. An example of a subject that is at particular riskof developing infection with a parasitic protozoan is a person travelingto an area in which infection with a parasitic protozoan is prevalent.Treatment can be performed before, during, or after the diagnosis ordevelopment of symptoms of infection. Treatment initiated after thedevelopment of symptoms may result in decreasing the severity of thesymptoms of one of the conditions, or completely removing the symptoms.A substituted aurone can be introduced into the mammal at any stage oftrypanosomal infection including, for example, during acute, earlycongenital, and/or reactivated trypanosomal infection.

Administration of a substituted aurone can occur before, during, and/orafter other treatments. Such combination therapy can involve theadministration of a substituted aurone during and/or after the use ofother anti-trypanosomal agents. The administration a substituted auronecan be separated in time from the administration of otheranti-trypanosomal agents by hours, days, or even weeks.

Methods to Treat or Prevent Fungal Disease or Infection

Substituted aurones including Aurone 1009 and/or Aurone 9051 can be usedto treat or prevent fungal infections, particularly fungal infectionscaused by yeast. Exemplary fungal infections include, but are notlimited to, an infection with a Candida species including, for example,C. albicans, C. tropicalis, C. stellatoidea, C. glabrata, C. krusei, C.parapsilosis, C. guilliermondii, C. visivanathii, or C. lusitaniae; aninfection with Rhodotorula mucilaginosa; an infection with aCryptococcus species, such as C. neoformans or C. gattii, and infectionwith a Saccharomyces species, such as S. cerevisiae, and infection witha Trichophyton species, such as T. rubrum. This disclosure provides atherapeutic method of treating a subject suffering from an infectionwith a fungus by administering a substituted aurone to the subject.Therapeutic treatment is initiated after diagnosis or the development ofsymptoms of infection with a fungus.

An aurone can also be administered prophylactically, to prevent or delaythe development of infection with a fungus. Treatment that isprophylactic, for instance, can be initiated before a subject manifestssymptoms of infection with a fungus. An example of a subject that is atparticular risk of developing infection with a fungus is animmunocompromised person. Treatment can be performed before, during, orafter the diagnosis or development of symptoms of infection. Treatmentinitiated after the development of symptoms may result in decreasing theseverity of the symptoms of one of the conditions, or completelyremoving the symptoms. An aurone can be introduced into the mammal atany stage of fungal infection.

Administration of an aurone can occur before, during, and/or after othertreatments. Such combination therapy can involve the administration ofan aurone during and/or after the use of other anti-fungal agents. Theadministration an aurone can be separated in time from theadministration of other anti-fungal agents by hours, days, or evenweeks.

Methods to Treat or Prevent Immune-Related Diseases, Disorders orConditions, Including Inflammation

Substituted aurones, including those having Formula II, as exemplifiedby Aurone 9067 (Aurone 1), can be sued to treat, prevent, inhibit, orcontrol inflammation and other immune-related conditions. Theimmune-related conditions can be chronic or acute; systemic orlocalized; autoimmune or associated with an infection caused by anexogenous agent. In one embodiment, an immunomodulatory aurone isadministered in an amount effective to treat or prevent inflammationand/or autoimmune disease. Administration of the composition can beperformed before, during, or after a subject develops an inflammatorycondition or autoimmune disease, or manifests inflammation or symptomsof inflammation or autoimmune disease. Therapeutic treatment isinitiated after the development of inflammation and/or autoimmunedisease. Treatment initiated after the development of an inflammatorycondition or autoimmune disease, or after manifestation of inflammationor symptoms of inflammation, may result in decreasing the severity ofthe symptoms of one of the conditions, or completely removing thesymptoms. In another embodiment, the immunomodulatory aurone isadministered prophylactically in an amount effective to prevent or delaythe development of inflammation and/or autoimmune disease in a subject.Treatment that is prophylactic, for instance, can be initiated before asubject develops an inflammatory condition or autoimmune disease, ormanifests inflammation or symptoms of inflammation or autoimmunedisease. An example of a subject who is at particular risk of developinginflammation or autoimmune disease is a person having a risk factor,such as a genetic marker, that is associated with inflammatory diseaseor autoimmune disease, or a person who has recently received atransplant. Another example is a subject who is suffering from a diseaseassociated with inflammation, but who has not developed an inflammatoryresponse.

Examples of diseases, disorders or conditions that can be treated orprevented by the composition of the invention include, withoutlimitation, rheumatoid arthritis (RA), inflammatory bowel disease (IBD)including Crohn's disease and ulcerative colitis, idiopathic orbitalinflammation, plaque psoriasis, psoriatic arthritis, ankylosingspondylitis, juvenile idiopathic arthritis, lupus, myasthenia gravis,focal segmental glomerulosclerosis, macrophage activation syndrome,non-Hodgkin's lymphoma, chronic lymphoid leukemia, precursorlymphoblastic lymphoma, familial Mediterranean fever (FMF), neonatalonset multisystem inflammatory disease (NOMID), tumor necrosis factorreceptor-associated periodic syndrome (TRAPS), deficiency of theinterleukin-1 receptor antagonist (DIRA), and Behçet's disease.

Additional inflammatory disorders that can be treated or prevented usingthe method of the invention include, for example, transplant rejection,graft vs. host disease, asthma, allergic reactions, chronic prostatitis,pelvic inflammatory disease, glomerulonephritis, reperfusion injury, andvasculitis; others include obesity, diabetes, infectious diseases,cancer, depression, heart disease, stroke, and Alzheimer's Disease.Diseases, conditions or disorders characterized by inflammation mayinclude the suffix “itis,” and it is expected that any disease,disorder, or condition having “itis” as part of its name can be treatedor prevented using the composition of the invention. Inflammation alsoplays an important role in the pathogenesis of atherosclerosis. The linkbetween rheumatoid arthritis and an increased risk of cardiovasculardisease and mortality is well established. Thus, an immunomodulatoryaurone is useful for treating cardiovascular disease associated with orcaused by other inflammatory conditions.

Administration of an immunomodulatory aurone can occur before, during,and/or after other treatments. Such combination therapy can involve theadministration of an immunomodulatory aurone before, during and/or afterthe use of other anti-inflammatory agents, for example, non-steroidalanti-inflammatory drugs, corticosteroids, blockers, and other activeagents as described herein for cumulative therapy or reduction orelimination of side effects. In a particularly preferred embodiment, theinvention contemplates combination therapy that employs, in addition toan immunomodulatory aurone, one or more immunomodulators and/or one ormore biologics to treat patients with autoimmune diseases such asrheumatoid arthritis (RA) and inflammatory bowel disease (IBD),including Crohn's disease and ulcerative colitis. The therapeutic andprophylactic methods of the invention therefore encompass administrationof a pharmaceutical composition that contains a first active agent thatincludes, as an immunomodulatory compound, an aurone or derivativethereof, and a second active agent that includes at least one of animmunomodulatory compound (in addition to the an immunomodulatoryaurone) and/or a biologic compound. The second active agent include oneor more compounds selected from, without limitation, azathioprine,6-mercaptopurine, cyclosporine A, tacrolimus, methotrexate,amethopterin, leflunomide, hydroxychloroquine, sulfasalazine,minocycline, prednisone, prednisolone, infliximab, adalimumab,etanercept, tocilizumab, certolizumab pegol, anakinra, abatacept,rituximab, and golimumab. Additional synthetic immunomodulatorycompounds and/or biologics that can serve as a second active agentinclude corticosteroids or glucocorticoids such as dexamethasone,sirolimus, mycophenolatmofetil, cyclophosphamide, daclizumab,basiliximab, antithymocyte globulin, muromunab, efalizumab, levamisole,recombinant cytokines such as aldesleukin, interferon-α, andinterferon-γ, and isoprinosine, See Jantan et al., Front Plant Sci 2015,6:655 (epub Aug. 25, 2015) for a description of exemplary syntheticimmunomodulatory agents.

Alternatively or additionally, a second active agent can include animmunomodulatory plant compound; or variant, derivative, analog,modification or conjugate thereof Δn exemplary list of such compoundscan be found in Jantan et al., Front Plant Sci 2015, 6:655 (epub Aug.25, 2015). Plant-derived immunomodulatory agents include alkaloids suchas berberine, chelerythrine, gelselegine, pseudocoptisine, leonurine,piperine, sinomenine, koumine, lycorine, sophocarpine, rhynchophylline,tetrandrine, and matrine; and essential oils such as Z-ligustilide andtetramethylpyra-zine. Plant-derived immunomodulatory flavonoids includechalcones such as butein, xanthohumol, dihydroxanthohumol,mallotophilippens C, D and E, and locochalcone E; flavones such asluteolin, apigenin, chrysin, nobiletin, baicalein, oroxylin A, andwogonin; flavonols such as quercetin, kaempferol, and rutin; flavanolssuch as epigallocatechin-3-gallate; isoflavones such as daidzein,genistein and puerarin; phioroglucinols such as myrtucommulone andarzanol; quinones such as thymoquinone, shikonin, and emodin-8-O-β-Dglucoside; stilbenes such as resveratrol and piceatannol; terpenoidssuch as 14-deoxyandrographolide,14-deoxy-11,12-didehydroandrographolide, ginsan, oleanolic acid,echinocystic acid, triptolide, demethylzelasteral, celastrol,asiaticoside, madecassoside, and 11-keto-β-boswellic acid; and apocynin.cis- or trans-Gnetin H can also be included as a second active agent.Plant-derived immunomodulatory compounds that have been the subject ofclinical trials include curcumin, resveratrol, epigallocatechin,quercetin, capsaicin, colchicine, andrographolide and genistein. Jantanet al., Front Plant Sci 2015, 6:655 (epub Aug. 25, 2015).

The administration of an immunomodulatory aurone can be separated intime from the administration of other active agents, such as additionalimmunomodulatory agents and/or biologics, by hours, days, or even weeks;alternatively, the other active agents can be administered concurrently,either together in the same composition or in separate compositions.Additionally or alternatively, the administration of an immunomodulatoryaurone can be combined with other biologically active agents ormodalities such as, for example, anti-inflammatory chemotherapeuticagents, and non-drug therapies, such as, but not limited to,radiotherapy, heat therapy, cryotherapy, electrical therapy, massage,and acupuncture.

Exemplary Treatment Populations

The compounds of the invention find utility in the treatment, control orprevention of trypanosomal or fungal infection and disease, or in thetreatment, control, or prevention of immune-related diseases, disorders,or conditions, not only in humans but also in animals. Compounds of theinvention can be administered to companion animals, domesticated animalssuch as farm animals, animals used for research, or animals in the wild.Companion animals include, but are not limited to, dogs, cats, hamsters,gerbils and guinea pigs. Domesticated animals include, but are notlimited to, cattle, horses, pigs, goats and llamas. Research animalsinclude, but are not limited to, mice, rats, dogs, apes, and monkeys. Inone embodiment, the compound of the invention is administered to ananimal, such as a companion animal or domesticated animal, that has beendiagnosed with, or is exhibiting symptoms of, or is at risk ofdeveloping, a trypanosomal or fungal infection. In another embodiment,the compound of the invention is administered in an animal or animalpopulation that serves, may serve, or is suspected of serving as atrypanosomatid or fungal reservoir, regardless of the presence ofsymptoms. Administration can be, for example, part of a small or largescale public health infection control program. The compound of theinvention can, for example, be added to animal feed as a prophylacticmeasure for reducing, controlling or eliminating trypanosomal or fungalinfection in a wild or domestic animal population. The compound can, forexample, be administered as part of routine or specialized veterinarytreatment of a companion or domesticated animal or animal population. Itshould be understood that administration of the compound of theinvention can be effective to reduce or eliminate trypanosomal or fungalinfection or the symptoms associated therewith; to halt or slow theprogression of infection or symptoms within a subject; and/or tocontrol, limit or prevent the spread of infection within a population;or movement of infection to another population.

Veterinary uses of the immunomodulatory aurones in domestic ordomesticated animals (including small animals such as cats, dogs, andother pets, as well as large animals such as cows, horses, pigs, andother livestock), as well as wild animals (e.g., animals housed in zoos)to treat or prevent inflammation or otherwise modulate an animal'simmune response, are examples of contemplated applications. Exemplarycompositions for veterinary use, such as vaccines, may contain, inaddition to an immunomodulatory aurone as described herein, routinevaccine components such as those included in vaccinations for distemper,rabies, feline leukemia, and other animal diseases, as well as othermedications, thereby allowing an immunomodulatory aurone or variant,derivative, analog, modification; or conjugate thereof to beco-administered with substances that might otherwise triggerinflammation, particularly in sensitive, diseased or vulnerable animals,such as the very young or very old.

Kits

The invention further includes a kit that contains at least one of asubstituted aurone or derivative thereof, together with instructions foruse. In some embodiments, the instructions for use provide instructionsfor use in the treatment or prevention of a trypanosomal infection ordisease, a fungal infection or disease, or inflammation or aninflammatory and/or autoimmune disease, disorder or condition.Optionally, the kit includes a pharmaceutically acceptable carrier. Thecarrier may be separately provided, or it may be present in acomposition that includes an substituted aurone or derivative thereof.Optionally, the kit may further include one or more additional activeagents which can be co-administered with the substituted aurone orderivative thereof. The one or more active agents may have cumulative orcomplementary activities, as described in more detail elsewhere herein.

Nutritional Supplement and Food Additive

A substituted aurone can be packaged as a nutritional, health or dietarysupplement (e. g., in pill or capsule form). The supplement can beoptionally formulated for sensitive populations, and thus can begluten-free, wheat-free, dairy-free, sugar-free and/or free ofpreservatives. Additionally, a substituted aurone can be added to a foodproduct to yield what is commonly referred to as a “nutriceutical” foodor “functional” food. Foods to which a substituted aurone can be addedinclude, without limitation, animal feed, cereals, yoghurts, cottagecheeses and other milk products, oils including hydrogenated orpartially hydrogenated oils, soups and beverages. Substituted auroneshaving one or more lipophilic or hydrophobic substitutions arepreferably incorporated into oily or fatty food products, to facilitatesolubilization. In one embodiment, a substituted is formulated as anutritional supplement or food additive for domestic or domesticatedanimals, such as pets or livestock. Conveniently, a substituted auroneor derivative thereof can be incorporated into animal feed such asfodder and kibble.

EXAMPLES

The invention is illustrated by the following examples. It is to beunderstood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example I. Synthesis of Aurone-Based Compounds

A library of over 100 aurone-based compounds was synthesized andevaluated for biological activity and mammalian cell toxicity.

Chemistry

Syntheses of the aurones used in this research were accomplished usingthe condensation approach between a coumaranone and an aldehyde (Scheme1):

Compound 6615 represents the basic, unsubstituted aurone Scaffold A inTable Example II).

Most of the aurone derivatives were prepared using the deep eutecticsolvent (DES conditions reported by Handy and Hawkins. It has recentlybeen observed that the purification of these products can be morerapidly and effectively accomplished via trituration with either etheror, in the case of particularly non-polar compounds, ether/hexanesmixtures. In addition to requiring less time than chromatography, theisolated yields are generally higher. For example, in the reaction ofthe unsubstituted coumaranone with 3-nitrobenzaldehyde, yields increasedfrom 40% to 75% by employing this change in purification with noreduction in compound purity. For compounds with free phenol groups, thetraditional potassium hydroxide in methanol conditions often workedbest. Most recently, we have noted that the combination of microwaveheating with the DES solvent dramatically increases the yield anddecreases the reaction time. For example, in the reaction with5-hydroxymethyl-2-furaldehyde, conventional heating afforded at besttraces of the desired aurone, while application of the microwave heatingconditions result in 28% of this sensitive product. It should be notedthat in this preliminary work, reactions have been optimized, andtherefore yields should be considered on the low end. The specificreaction conditions employed for the synthesis of representative auronesare noted in the experimental section of this paper. Structures forrepresentative aurone compounds are shown below, and in Table 1 (ExampleII).

Table 1 (Example II) shows the various scaffolds used in the chemicalsyntheses of exemplary substituted aurones, as well as selectedbiological data, such as anti-trypanosomal activity, for exemplarysubstituted aurones.

Materials and Methods

All compounds have been synthesized as described below and characterizedwith respect to identity and purity using NMR, IR, and melting point.The thermodynamically favored Z isomer is assumed to be the majorproduct in accordance with the literature in all cases, unless specifiedotherwise.

Compounds 2001-2025 have been previously published by Hawkins and Handy(Hawkins et al., Tetrahedron 2013, 69, 9200-9204), and were prepared asdescribed therein. Sample IDs in this and the following examples have insome cases been recoded as follows: 2001=A, 2002=B, 2004=D, 2008=H,2009=1, 2010=J, 2011=K, 2013=M, 2014=N, 2015=O, 2018=R, 2021=U, 2023=W,2026=Z.

Method #1—Handy Traditional (Hawkins et al., Tetrahedron 2013, 69,9200-9204)

In the following syntheses, coumaranone (1.00 mmol) and aldehyde (1.00mmol) were combined in a dry vial and 1 mL of the deep eutectic solvent(DES) formed from a 1:2 molar ratio of choline chloride and urea wasadded. The reaction mixture was heated to 80° C. and stirred for 12hours. At this point, the reaction was cooled to room temperature andpartitioned between water and methylene chloride. The organic layer wasseparated and concentrated to dryness in vacuo to afford the desiredaurone. Further purification was performed as noted.

1. (Z)-2-((1H-imidazol-2-yl)methylene)benzofuran-3(2H)-one (2026)

(CDCl₃, 300 MHz): 7.80 (s, 1H), 7.72 (s, 1H), 7.35 (d, J=6.5 Hz, 1H),7.18 (d, J=6.5 Hz, 1H), 6.94 (t, 1H), 6.79 (d, J=6.0 Hz, 1H), 6.58 (d,J=6.0 Hz, 1H), 6.38 (t, 1H)

2. (Z)-6,7-dihydroxy-2-(thiophen-2-ylmethylene)benzofuran-3(2H)-one(1001)

This reaction was performed on a 0.5 mmol scale. The crude solid waspurified by washing with diethyl ether to yield 5.6 mg (2.15%) of 1001as an orange-red solid (MP=168-170° C.). IR (neat, thin film):3100-3500, 2500, 1700, 1425 cm⁻¹; ¹H NMR (DMSO, 300 MHz) 7.85 (d, J=5.16Hz, 1H), 7.71 (d, J=3.45 Hz, 1H), 7.18 (dd, J1,3=8.58 Hz, J1,2=3.6 Hz1H), 7.10 (s, 1H), 7.07 (d, J=3.0 Hz, 1H), 6.60 (d, J=8.4 Hz, 1H); ¹³CNMR (DMSO, 75 MHz) 181.085, 160.034, 154.4924, 154.255, 146.929,135.778, 133.108, 131.992, 130.694, 128.672, 116.349, 113.611, 104.216.

3. (Z)-6-hydroxy-2-(thiophen-2-ylmethylene)benzofuran-3(2H)-one (2901)

This reaction was performed on a 0.5 mmol scale. The product wasinsoluble in dichloromethane and precipitated out of to yield 82.3 mg(67.38%) of 2901 as a yellow-brown solid (MP=300-303° C.). IR (neat,thin film): 3080, 2920, 1700, 1630, 1570, 1460, 1320, 1280, 1130, 1110cm⁻¹; ¹H NMR (DMSO D₆, 300 MHz) 7.859 (d, J=4.8 Hz, 1H), 7.649 (d,J=3.45 Hz, 1H), 7.570 (d, J=8.25 Hz, 1H), 7.189 (d, J=4.47 Hz, 1H),7.160 (s, 1H), 6.728 (s, 1H), 6.680 (d, J=8.25 Hz, 1H); ¹³C NMR (DMSO,75 MHz) 181.1441, 167.8997, 167.1444, 146.0632, 135.4317, 134.0359,132.8695, 128.7393, 126.4352, 113.8820, 113.6908, 105.6312, 99.0726.

4. (Z)-6-methoxy-2-(thiophen-2-ylmethylene)benzofuran-3(2H)-one (5001)

This reaction was performed on a 0.5 mmol scale. The crude solid waspurified by washing with diethyl ether to yield 123.8 mg (95.86%) of5001 as a brown solid (MP=99-103° C.). IR (neat, thin film) 3755, 1555,1440, 1310, 820, 690 cm¹; ¹H NMR (CDCl₃, 300 MHz): 7.66 ppm (d, J=8.25Hz, 1H), 7.55 ppm (d, J=5.13 Hz, 1H), 7.48 ppm (d, J=3.09 Hz, 1H), 7.12ppm (t, J=5.16 Hz, 1H), 7.08 ppm (s, 1H), 6.77 ppm (s, 1H), 6.72 ppm (d,J=12.36 Hz, 1H), 3.89 ppm (s, 3H) ¹³C NMR (CDCl₃, 75 MHz): 182.17,168.01, 167.31, 146.21, 135.54, 132.61, 131.20, 127.93, 125.63, 115.35,112.28, 105.92, 96.66, 56.03.

5. (Z)-6-methoxy-2-(4-methylbenzylidene)benzofuran-3(2H)-one (5002)

This reaction was performed on a 0.5 mmol scale. The crude solid waspurified by washing with diethyl ether to yield 83.6 mg (62.79%) of 5002as a tan solid (MP=115-122° C.). IR (neat, thin film): 3730, 2960, 2860,1640, 1390, 660 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.81 (d, J=7.92, 2H),7.72 (d, J=8.58 Hz, 2H), 7.24 (d, J=4.8 Hz, 1H), 6.78 (s, 1H), 6.82 (s,1H), 6.75 (d, J=3.45 Hz, 1H), 3.91 (s, 3H), 2.38 (s, 3H); ¹³C NMR(CDCl₃, 75 MHz): 183.16, 167.35, 147.51, 140.31, 131.45, 129.74, 129.25,125.88, 115.02, 114.85, 112.32, 112.22, 96.70, 56.01, 21.59.

6. (Z)-2-(4-bromobenzylidene)-6-methoxybenzofuran-3(2H)-one (5005)

This reaction was performed on a 0.5 mmol scale. The crude solid waspurified by washing with diethyl ether to yield 186 mg (56.18%) of 5005as a brown solid (MP=153° C.). IR (neat, thin film): 3300, 1670, 1600,1480, 1280, 1010, 950, 820, 620 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.74 (d,J=8.58 Hz, 1H), 7.72 (s, 1H), 7.70 (d, J=2.34 Hz, 1H), 7.56 (d, J=8.58Hz, 2H), 6.78 (s, 1H), 6.72 (d, J=5.16 Hz, 2H), 3.94 (s, 3H); ¹³C NMR(CDCl₃, 75 MHz): 182.80, 167.60, 148.05, 132.59, 131.33, 125.93, 123.96,114.65, 112.36, 110.48, 96.69, 56.08.

7. (Z)-5-bromo-2-(4-methylbenzylidene)benzofuran-3(2H)-one (6000)

The crude solid was purified by via column chromatography using 5%EtOAc/Hexanes as the eluent to yield 156.7 mg (49.72%) of 6000 as ayellow solid (MP=115-120° C.). IR (neat, thin film): 2973, 2923, 2870,1705, 1642, 1593 cm⁻¹; 1HNMR (Acetone D₆. 500 MHz): 7.91 (m, 4H), 7.51(d, J=8.6 Hz, 1H), 7.34 (d, J=8.0 Hz, 2H), 2.40 (s, 3H). 6.90 (s, 1H);¹³CNMR (CDCl₃, 125 MHz): 183.05, 165.55, 147.28, 141.75, 140.31, 132.58,130.59, 130.25, 127.44, 124.19, 116.75, 116.21, 114.11, 21.58.

8. (Z)-5-bromo-2-(4-(trifluoromethyl)benzylidene)benzofuran-3(2H)-one(6001)

The crude solid was purified by washing with 10% Diethyl Ether/Hexanesto yield 83.7 mg (22.70%) of SH6001 as a reddish-brown solid(MP=120-125° C.). IR (neat, thin film): 3020, 2850, 1700, 1650 cm⁻¹;¹HNMR (CDCl₃, 500 MHz): 7.95 ppm (d, J=8.05 Hz, 2H), 7.87 ppm (s, 1H),7.73 ppm (d, J=9.2 Hz, 1H), 7.66 ppm (d, J=8 Hz, 2H), 7.21 ppm (d,J=8.45 Hz, 1H), 6.84 ppm (s, 1H); ¹³CNMR (CDCl₃, 75 MHz): 183.28,164.91, 147.57, 139.91, 131.64, 127.54, 125.90, 125.76, 123.13, 122.99,116.81, 114.79, 111.89, 111.18.

9. (Z)-5-bromo-2-(4-bromobenzylidene)benzofuran-3(2H)-one (6002)

The crude solid was purified by washing with diethyl ether to yield 94.2mg (27.5,%) of 6002 as a yellow solid (MP=126-130° C.). IR (neat, thinfilm): 3100, 1700, 1650, 1600, 1420, 1280, 1180, 1080, 1010, 805 cm⁻¹;¹H NMR (CDCl₃, 300 MHz) 7.89 (d, J=2.07 Hz, 1H), 7.72 (dd, J=8.9, 2.2Hz, 2H), 7.55 (d, J=8.58 Hz, 2H), 7.22 (d, J=8.94 Hz, 2H) 6.81 (s, 1H);¹³C NMR (CDCl₃, 75 MHz) 183.11, 164.61, 146.92, 139.54, 132.89, 132.23,130.80, 127.39, 124.77, 123.17, 116.54, 114.68, 112.70.

10. (Z)-4-((5-bromo-3-oxobenzofuran-2(3H)-ylidene)methyl)benzonitrile(6003)

The crude solid was purified by washing with diethyl ether to yield 67.8mg (20.79%) of 6003 as a yellow solid (Decomp=170° C.). IR (neat, thinfilm): 3000, 2200, 1600-1700 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.97 (m,2H), 7.91 (d, J=1.71 Hz, 1H), 7.76 (dd, J=8.90, 2.43 Hz, 1H), 7.73 (d,J=8.58 Hz, 2H), 7.25 (t, J=4.11 Hz, 1H), 6.83 (d, J=6.54 Hz, 1H); ¹³CNMR (125 MHz, CDCl₃) 183.07, 164.86, 147.97, 139.83, 136.34, 132.47,131.65, 127.61, 124.96, 124.08, 116.98, 114.74, 112.74, 111.95, 109.95.

11. (Z)-4,6 dimethoxy-2-(4-methylbenzalidene) benzofuran-3(2H)-one(7000)

The crude solid was purified by washing with ethyl acetate to yield 92.0mg (31.05%) of 7000 as a yellow solid (MP=145-148° C.). IR (neat, thinfilm): 3423, 3407, 1691, 1615, 1505, 1461, 1436, 1344, 1247, 1212, 1156,1093, 1043, 946, 812, 752, 697 cm⁻¹; ¹H NMR (DMSO-D₆, 300 MHz) 7.79 (d,J=8.2 Hz, 2H), 7.27 (d, J=8.1 Hz, 2H), 6.69 (d, J=1.8 Hz, 1H), 6.65 (s,1H), 6.31 (d, J=1.8 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H), 2.32 (s, 3H);¹³C NMR (DMSO-D₆, 75 MHz) 179.52, 169.44, 168.72, 159.42, 147.43,140.09, 131.45, 130.14, 129.88, 110.18, 104.58, 94.97, 90.43, 57.07,56.68, 21.66.

12. (Z)-4,6-dimethoxy-2-(4-nitrobenzylidene)benzofuran-3(2H)-one (7001)

The crude solid was insoluble in EtOAc and precipitated to yield 8.2 mg(2.51%) of 7001 as an orange solid (Decomp at 180° C.). IR (neat, thinfilm): 2980, 1700, 1600, 1520, 1340, 1220, 1160, 1090, 810 cm⁻¹; 1H NMR(DMSO D₆, 300 MHz): 8.71 (s, 1H), 8.31 (d, J=7.56 Hz, 1H), 8.21 (d,J=6.54 Hz, 1H), 7.73 (t, J=7.93, 1H), 6.87 (s, 1H), 6.69 (s, 1H), 6.34(s, 1H), 3.91 (s, 3H), 3.87 (s, 3H).

13. (Z)-2-(4-ethylbenzylidene)benzofuran-3(2H)-one (8001)

The crude solid was purified by via column chromatography using 10%EtOAc/Hexanes as the elutent on neutral alumina to yield 56.6 mg(22.61%) of 8001 as a yellow-orange solid (MP=71-74° C.). IR (neat, thinfilm): 3060, 2980, 1710, 1650, 1600, 1480, 1300, 1200, 1100, 890, 750cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.85 (d, J=8.2 Hz, 2H), 7.81 (ddd, J=7.6,1.4, 0.6 Hz, 1H), 7.65 (ddd, J=8.7, 7.3, 1.4 Hz, 1H), 7.31 (m, 3H), 7.22(td, J=7.7, 0.8 Hz, 1H), 6.90 (s, 1H), 2.70 (q, J=7.6 Hz, 2H), 1.27 (t,J=7.6 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): 184.80, 166.09, 146.84, 146.62,136.82, 131.79, 129.83, 128.60, 124.66, 123.44, 121.85, 113.46, 113.02,29.03, 15.40.

14. (Z)-2-(4-isopropylbenzylidene)benzofuran-3(2H)-one (8002)

The crude solid was purified by via column chromatography using 1%EtOAc/Hexanes as the elutent on silica to yield 114.9 mg of 8002 as ayellow oil. IR (neat, thin film): 3300 (br), 2920, 1700, 1650, 1600,1460, 1300, 1100, 880, 750 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.87 (d, J=8.3Hz, 1H), 7.82 (dt, J=7.8, 0.6 Hz, 1H), 7.65 (m, 1H), 7.33 (dd, J=8.4,0.5 Hz, 2H), 7.22 (t, J=7.5 Hz, 1H), 6.91 (s, 1H), 2.96 (dt, J=13.8, 6.9Hz, 1H), 1.28 (d, J=6.9 Hz, 3H); ¹³C NMR (CDCl₃, 75 MHz): 184.88,166.15, 151.45, 146.68, 136.84, 131.82, 129.99, 127.20, 124.72, 123.45,121.89, 113.48, 113.03, 34.29, 23.85.

15. (Z)-2-(4-fluorobenzylidene)benzofuran-3(2H)-one (9002)

The crude solid was purified by washing with diethyl ether to yield 9.8mg (4.29%) of 9002 as a yellow solid (MP=144-147° C.). IR (neat, thinfilm): 3030, 2920, 1710, 1650, 1600, 1500, 1220, 1120, 890, 850, 750cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.93 (m, 2H), 7.81 (ddd, J_(1,5)=7.8 Hz,J_(1,3)=1.5 Hz, J_(1,2)=0.6 Hz, 1H), 7.67 (ddd, J_(1,4=8.4) Hz,J_(1,3)=7.2 Hz, J_(1,2)=1.5 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H), 7.23 (t,J=7.8 Hz, 1H), 7.15 (m, 2H), 6.86 (s, 1H); ¹³C NMR (CDCl₃, 75 MHz):184.81, 166.19, 165.20, 161.15, 146.64, 137.07, 133.64 (d, J=33 Hz, 2C),128.68 (d, J=1.5 Hz), 124.12 (d, J=292 Hz), 121.71, 116.24 (d, J=87 Hz,2C), 113.02, 111.97

16. (Z)-2-(2-iodobenzylidene)benzofuran-3(2H)-one (9003)

The crude solid was purified by washing with diethyl ether to yield146.18 mg (41.99%) of 9003 as a yellow solid (MP=145-148° C.). IR (neat,thin film): 3061, 1702, 1651, 1597, 1450, 1299, 1185, 1099, 952, 885,746 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 8.28 (dd, J=8.0, 1.6 Hz, 1H), 7.96(dd, J=8.0, 1.2 Hz, 1H), 7.83 (ddd, J=8.2, 1.1, 0.4 Hz, 1H), 7.66 (ddd,J=8.5, 7.4, 1.3 Hz, 1H), 7.46 (m, 1H), 7.31 (d, J=8.3 Hz, 1H), 7.22 (t,J=6.9 Hz, 1H), 7.18 (s, 1H), 7.05 (td, J=7.9, 1.6 Hz, 1H); ¹³C NMR(CDCl₃, 70 MHz): 185.954, 167.608, 148.859, 141.539, 138.572, 136.665,133.196, 132.233, 129.873, 126.235, 125.093, 122.856, 117.179, 114.328,104.251.

17. (Z)-2-(2-fluorobenzylidene)benzofuran-3(2H)-one (9004)

The crude solid was purified by washing with diethyl ether to yield111.7 mg (46.49%) of 9004 as an orange-yellow solid (MP=76-81° C.). IR(neat, thin film): 3020, 2920, 1710, 1610, 1150, 750 cm⁻¹: ¹H NMR(CDCl₃, 500 MHz): 8.32 (td, J=7.7, 1.7 Hz, 1H), 7.81 (ddd, J=7.5, 1.5,0.5 Hz, 1H), 7.66 (ddd, J=8.6, 7.4, 1.4 Hz, 1H), 7.37 (m, 1H), 7.32 (d,J=8.3 Hz, 1H), 7.26 (m, 1H), 7.23 (t, J=7.5 Hz, 1H), 7.19 (s, 1H), 7.12(ddd, J=10.0, 8.5, 1.5 Hz, 1H).

18. (Z)-2-(2-bromobenzylidene)benzofuran-3(2H)-one (9006)

The crude solid was purified by washing with diethyl ether to yield151.0 mg (50.14%) of 9006 as a yellow solid (MP=134-140° C.). IR (neat,thin film): 2980, 1700, 1600, 1450, 780 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz):8.34 (dd, J=7.9, 1.6 Hz, 1H), 7.82 (ddd, J=7.6, 1.4, 0.6 Hz, 1H), 7.67(m, 2H), 7.43 (m, 1H), 7.31 (d, J=7.0 Hz, 2H), 7.23 (m, 2H). ¹³C NMR(CDCl₃, 125 MHz): 184.643, 166.261, 147.623, 137.216, 133.481, 132.454,132.110, 130.856, 127.766, 126.643, 124.931, 123.810, 121.538, 113.027,110.833.

19. (Z)-2-(2-chlorobenzylidene)benzofuran-3(2H)-one (9007)

The crude solid was purified by washing with diethyl ether to yield 62.0mg (24.17%) of 9007 as a yellow solid (MP=118-126° C.). IR (neat, thinfilm): 2940, 1700, 1650, 1600, 1300, 1180, 1110, 890 cm⁻¹; ¹H NMR(CDCl₃, 300 MHz): 8.36 (dd, J=7.8 Hz, J=1.8 Hz, 1H), 7.83 (ddd, J=7.5Hz, J=1.2 Hz, J=0.6 Hz, 1H), 7.67 (ddd, J=8.4 Hz, J=7.2 Hz, J=1.5 Hz,1H), 7.47 (dd, J=7.8 Hz, J=1.5 Hz, 1H), 7.32 (m, 5H); ¹³C NMR (CDCl₃, 75MHz): 184.68, 166.26, 147.68, 137.20, 136.04, 132.34, 130.71, 130.47,130.11, 127.16, 124.94, 123.80, 121.57, 113.03, 108.12.

20. (Z)-2-(4-chlorobenzylidene)benzofuran-3(2H)-one (9019)

The crude solid was purified by washing with diethyl ether to yield 70.1mg (27.32%) of 9019 as a yellow solid (MP=138-145° C.). IR (neat, thinfilm): 2980, 1700, 1610, 1300, 860 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.84(d, J=8.5 Hz, 2H), 7.80 (dd, J=7.6 Hz, J=0.7 Hz, 1H), 7.66 (ddd, J=8.6,J=7.4 Hz, 1J=0.4 Hz, 1H), 7.41 (d, J=8.6 Hz, 2H), 7.33 (d, J=8.3 Hz,1H), 7.23 (t, J=7.1 Hz, 1H), 6.82 (s, 1H); ¹³C NMR (CDCl₃, 75 MHz):184.75, 166.17, 147.07, 137.17, 135.95, 132.72, 130.88, 129.30, 124.85,123.76, 121.59, 113.04, 111.68.

21. (Z)-2-(3-fluorobenzylidene)benzofuran-3(2H)-one (9024)

The crude solid was purified by washing with diethyl ether to yield 86.9mg (36.18%) of 9024 as a yellow solid (MP=111-113° C.). IR (neat, thinfilm): 3030, 1710, 1660, 1600, 1450, 1300, 1140, 890, 750, 680 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz): 7.80 (dd, J=7.7, 0.8 Hz, 1H), 7.69 (m, 2H), 7.59(d, J=7.8 Hz, 1H), 7.40 (td, J=8.0, 6.0 Hz, 1H), 7.34 (d, J=8.3 Hz, 1H),7.23 (m, 1H), 7.10 (tdd, J=8.4, 2.6, 0.9 Hz, 1H), 6.83 (s, 1H). ¹³C NMR(CDCl₃, 75 MHz): 184.67, 166.15, 164.46, 161.20, 147.24, 137.16, 134.28(d, J=33 Hz), 130.26 (d, J=33 Hz), 127.40 (d, J=1.2 Hz), 124.02 (d, 289Hz), 121.37, 117.50 (d, J=87 Hz), 116.79 (d, J=90 Hz), 112.98, 111.39(d, J=1.2 Hz).

22. (Z)-2-(3-chlorobenzylidene)benzofuran-3(2H)-one (9026)

The crude solid was purified by washing with diethyl ether to yield 37.8mg (14.72%) of 9026 as a yellow solid (MP=86-89° C.). IR (neat, thinfilm): 3030, 2920, 1710, 1600, 1400, 1100, 750 cm⁻¹: ¹H NMR (CDCl₃, 300MHz): 7.91 (s, 1H), 7.78 (ddd, J=7.6 Hz, J=1.4 Hz, J=0.6 Hz, 1H), 7.72(m, 1H), 7.65 (ddd, J=8.4 Hz, J=7.2 Hz, J=1.5 Hz, 1H), 7.35 (m, 3H),7.21 (ddd, J=8.1 Hz, J=7.5 Hz, J=0.8 Hz, 1H), 6.77 (s, 1H); ¹³C NMR(CDCl₃, 75 MHz): 184.75, 166.26, 147.37, 137.31, 134.88, 134.09, 130.97,130.15, 129.84, 129.69, 124.85, 123.83, 121.44, 113.12, 111.26.

23. (Z)-2-(3-iodobenzylidene)benzofuran-3(2H)-one (9028)

The crude solid was purified by washing with diethyl ether to yield 69.2mg (19.88%) of 9028 as a yellow solid (MP=99-105° C.). IR (neat, thinfilm): 3060, 1700, 1610, 1490, 1320, 1200, 750 cm⁻¹; ¹H NMR (CDCl₃, 300MHz): 8.25 (s, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.78 (ddd, J=7.6 Hz, J=1.4Hz, J=0.6 Hz, 1H), 7.67 (m, 2H), 7.33 (d, J=8.3 Hz, 1H), 7.18 (m, 2H),6.73 (s, 1H); ¹³C NMR (CDCl₃, 75 MHz): 184.65, 166.25, 147.30, 139.90,138.62, 137.23, 134.50, 130.61, 130.52, 124.85, 123.81, 121.48, 113.13,111.04, 94.76.

24. (Z)-2-(4-iodobenzylidene)benzofuran-3(2H)-one (9029)

The crude solid was purified by washing with diethyl ether to yield 44.1mg (12.67%) of 9029 as a yellow-orange solid (MP=176-179° C.). IR (neat,thin film): 2980, 1700, 1650, 1600, 1450, 1300, 1200, 890 cm⁻¹; ¹H NMR(CDCl₃, 300 MHz) 7.80 (m, 3H), 7.64 (m, 3H), 7.33 (dt, J=8.4 Hz, J=0.7Hz, 1H), 7.23 (m, 1H), 6.79 (s, 1H); ¹³C NMR (CDCl₃, 75 MHz): 184.756,166.174, 147.349, 138.257, 137.194, 132.922, 131.823, 124.863, 123.780,121.584, 113.057, 111.833, 96.569.

25. (Z)-2-(3-bromobenzylidene)benzofuran-3(2H)-one (9030)

The crude solid was purified by washing with diethyl ether to yield 67.4mg (22.37%) of 9030 as a solid. IR (neat, thin film): ¹H NMR (CDCl₃, 500MHz): 8.09 (s, 1H), 7.80 (t, J=7.1 Hz, 2H), 7.67 (m, 1H), 7.51 (m, 1H),7.36 (d, J=8.6 Hz, 1H), 7.32 (t, J=7.9 Hz, 1H), 7.24 (t, J=7.5 Hz, 2H),6.78 (s, 1H). ¹³C NMR (CDCl₃, 125 MHz): 184.75, 166.29, 147.40, 137.29,134.43, 133.91, 132.73, 130.42, 130.10, 124.88, 123.84, 123.05, 121.48,113.14, 111.14.

26. (Z)-4-((6-hydroxy-3-oxobenzofuran-2(3H)-ylidene)methyl)benzonitrile(9076)

The crude solid was purified by washing with diethyl ether to yield147.7 mg (56.10%) of 9076 as a brown solid (MP=112-115° C.). IR (neat,thin film): 3500-3100, 1700, 1560 cm⁻¹; ¹H NMR (DMSO-D₆, 300 MHz) 8.07(d, J=8.4 Hz, 1H), 7.91 (d, J=8.5 Hz, 1H), 7.57 (d, J=8.5 Hz, 1H), 6.79(s, 1H), 6.69 (m, 1H), 6.63 (dt, J=4.1, 1.8 Hz, 1H).

28. (Z)-2-(4-(trifluoromethyl)benzylidene)benzofuran-3(2H)-one (9084)

The crude solid was purified by washing with diethyl ether to yield278.2 mg (95.9%) of 9084 as an orange-yellow solid (MP=98-102C). IR(neat, thin film): 3020, 1700, 1600, 1320, 1110, 1080, 750 cm¹: ¹H NMR(CDCl₃, 500 MHz): 7.96 (d, J=8.0 Hz, 2H), 7.77 (d, J=8.0 Hz, 1H), 7.64(m, 3H), 7.30 (d, J=8.5 Hz, 1H), 7.20 (t, 7.5 Hz, 1H), 6.82 (s, 1H); ¹³CNMR (CDCl₃, 125 MHz) 184.76, 166.32, 147.80, 137.48, 135.74, 131.50,131.00 (q, J=32 Hz), 129.03, 125.78 (q, J=4 Hz), 123.52 (q, J=270 Hz),122.17, 121.27, 113.06, 110.82.

29. (Z)-2-(3-(trifluoromethyl)benzylidene)benzofuran-3(2H)-one (9085)

The crude solid was purified by washing with diethyl ether to yield 236mg (81.4%) of 9085 as a yellow solid (MP=128-133° C.). IR (neat, thinfilm): 3020, 1700, 1650, 1600, 1110, 750, 690 cm⁻¹: ¹H NMR (CDCl₃, 300MHz): 8.15 (s, 1H), 8.03 (d, J=4.5 Hz, 1H), 7.78 (dd, J=4.8 Hz,J_(1,2)=0.6 Hz, 1H), 7.65 (ddd, J_(1,4)=5.1 Hz, J_(1,3)=4.5 Hz,J_(1,2)=0.9 Hz, 1H), 7.60 (d, J=4.8 Hz, 1H), 7.54 (t, J=4.8 Hz, 1H),7.33 (d, J=5.1 Hz, 1H), 7.21 (t, J=4.5 Hz, 1H), 6.84 (s, 1H); ¹³C NMR(CDCl₃, 75 MHz): 184.74, 166.31, 147.58, 137.40, 134.43, 133.15, 131.69,131.25, 129.47, 127.89, 127.84, 126.23, 126.16, 125.75, 124.91, 123.94,122.15, 121.39, 113.15, 110.93.

30. (Z)-2-(2-(trifluoromethyl)benzylidene)benzofuran-3(2H)-one (9086)

The crude solid was purified by washing with diethyl ether to yield 94.2mg (32.46%) of 9086 as a yellow solid (MP=118-123° C.). IR (neat, thinfilm): 2980, 2860, 1700, 1660, 1320, 1050 cm⁻¹; ¹H NMR (CDCl₃, 300 MHZ):8.40 (d, J=7.9 Hz, 1H), 7.83 (d, J=7.7 Hz, 1H), 7.75 (d, J=7.9 Hz, 1H),7.67 (t, J=7.4 Hz, 2H), 7.48 (t, J=7.6 Hz, 1H), 7.31 (d, J=8.3 Hz, 1H),7.24 (m, 1H), 7.19 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz): 207.73, 207.70,184.82, 166.47, 147.83, 137.42, 132.61, 132.02, 132.00, 130.39, 130.36,130.07, 129.66, 129.18, 126.48, 126.41, 126.33, 126.26, 125.83, 125.08,123.96, 122.21, 121.45, 113.04, 107.28, 107.25, 107.22, 107.18.

31. (Z)-2-(4-(dimethylamino)benzylidene)benzofuran-3(2H)-one (9087)

The crude solid was purified by washing with diethyl ether to yield 70.2mg (29.83%) of 9087 as a red solid (MP=168-170° C.). IR (neat, thinfilm): 3020, 1700, 1650, 1600, 1110, 750, 690 cm⁻¹; ¹H NMR (CDCl₃, 300MHz): 7.85 (d, J=5.4 Hz, 2H), 7.80 (d, J=3.9 Hz, 1H), 7.60 (t, J=4.2 Hz,1H), 7.31 (d, J=5.1 Hz, 1H), 7.18 (t, J=4.5 Hz, 1H), 6.92 (s, 1H), 6.75(d, J=5.4 Hz, 2H), 3.07 (s, 6H); ¹³C NMR (CDCl₃, 75 MHz): 184.09,165.36, 151.43, 145.10, 135.92, 133.74, 124.40, 122.97, 122.54, 120.07,115.46, 112.86, 112.02, 40.16.

32. (Z)-2-(2-hydroxybenzylidene)benzofuran-3(2H)-one (9088)

The crude solid was purified by washing with diethyl ether to yield120.1 mg (50.41%) of 9088 as a yellow solid (MP=215-216° C.). IR (neat,thin film): 3200 (br), 3100, 1710, 1650, 1600, 1450, 1380, 1120, 890,750 cm⁻¹; ¹H NMR (Acetone D₆, 300 MHz): 8.25 (dd, J=7.9, 1.6 Hz, 1H),7.76 (m, 2H), 7.48 (d, J=9.4 Hz, 1H), 7.39 (s, 1H) 7.28 (m, 2H), 7.02(d, J=8.4 Hz, 1H), 6.97 (t, J=7.6 Hz, 1H). ¹³C (Acetone-D₆, 75 MHz):183.42, 165.74, 159.48, 146.11, 136.79, 131.84, 131.60, 124.05, 123.56,121.86, 119.54, 118.87, 116.50, 113.09, 107.61.

Method #2—Varma (Varma et al., Tetrahedron Letters. 1992, 17, 5937-5940)

In the following syntheses, coumaranone (1.00 mmol) and aldehyde (1.00mmol) were combined in a dry vial. 3.5 g of neutral alumina was thenadded followed by 5 mL of dichloromethane. The reaction mixture wasstirred for 12 hours at 25° C. The reaction mixture was then filteredand the dichloromethane layer collected and concentrated to dryness invacuo to afford the desired aurone. Further purification was performedas noted.

0. (Z)-2-((1H-imidazol-2-yl)methylene)benzofuran-3(2H)-one (2026)(Alternative Synthesis)

The crude reaction mixture was then purified by trituration with etherto afford 24.0 mg (11%) of aurone 2026 as a brown solid (Decomp.=92-94°C.). IR (neat, thin film): 2950, 2820, 1700, 1610, 1500, 1310, 1090,750; 1H NMR (CDCl3, 300 MHz) δ 9.77 (s, 1H), 7.83 (dt, J=9.0, 1.4 Hz,1H), 7.68 (dq, J=9.0, 1.4 Hz, 1H), 7.38-7.21 (m, 4H), 7.06 (s, 1H). 13CNMR (CDCl3, 75 MHz): 183.34, 181.22, 165.47, 165.17, 147.72, 146.48,141.18, 137.60, 137.08, 124.92, 124.82, 124.11, 123.31, 121.79, 113.01,112.68, 112.41, 102.14.

1. Methyl (Z)-4-((3-oxobenzofuran-2(3H)-ylidene)methyl)benzoate (9047)

The crude solid was purified by washing with diethyl ether to yield104.7 mg (37.36%) of 9047 as a yellow solid (MP=144-147° C.). IR (neat,thin film): 2980, 1700, 1650, 1280, 1050, 1020 cm¹; ¹H NMR (CDCl₃, 300MHz): 8.10 (d, J=8.5 Hz, 2H), 7.96 (d, J=8.3 Hz, 2H), 7.81 (ddd, J=7.7Hz, J=1.4 Hz, J=0.6 Hz, 1H), 7.68 (ddd, J=8.6 Hz, J=7.3 Hz, J=1.4 Hz,1H), 7.35 (d, J=8.3 Hz, 1H), 7.24 (td, J=7.7 Hz, J=0.8 Hz, 1H), 6.87 (s,1H), 3.94 (s, 3H). ¹³C NMR (CDCl₃, 75 MHz): 184.85, 166.63, 166.36,147.82, 137.38, 136.71, 131.29, 130.69, 130.06, 124.93, 123.91, 121.42,113.12, 111.36, 52.34.

2. (Z)-2-((4,5-dibromothiophen-2-yl)methylene)benzofuran-3(2H)-one(9050)

The resulting product was purified through ether wash and yielded 177.6mg (46.00%) of aurone 9050 as a yellow-orange solid (MP=185-190° C.). IR(neat, thin film) 3600-3200, 2360, 1640, 1480, 1320 cm⁻¹; ¹H NMR (CDCl3,500 MHz) 7.791 (d, J=9.0 Hz, 1H), 7.673 (t, J=12.0 Hz, 1H), 7.338 (d,J=8.0 Hz, 1H), 7.281 (s, 1H), (t, J=7.5 Hz, 1H), 6.940 (s, 1H); ¹³C NMR(CDCl3, 75 MHz) 183.59, 165.75, 146.15, 137.14, 136.93, 134.07, 124.84,123.95, 121.96, 117.69, 115.35, 113.12, 104.79.

3. (Z)-2-(pyridin-2-ylmethylene)benzofuran-3(2H)-one (9051)

The resulting product was purified through ether wash and yielded 16.92mg (7.58%) of aurone 9051 as a brown solid (MP=119-121° C.). IR (neat,thin film): 3060, 1710, 1660, 1610, 1480, 1310, 1200, 1150, 890 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz): 8.72 (d, J=4.0 Hz, 1H), 8.15 (d, J=8.0 Hz, 1H),7.79 (m, 2H), 7.66 (m, 1H), 7.34 (d, J=8.3 Hz, 1H), 7.24 (m, 2H), 7.02(s, 1H). ¹³C NMR (CDCl₃, 75 MHz): 184.95, 166.54, 152.06, 150.41,148.14, 137.38, 136.56, 126.77, 124.92, 123.91, 123.39, 121.38, 113.17,112.58.

4. (Z)-2-(4-hydroxy-3-methoxybenzylidene)benzofuran-3(2H)-one (9053)

The resulting product was purified through ether wash and yielded 82.79mg (30.86%) of aurone 9053 as an orange solid (Decomp at 185° C.). IR(neat, thin film): 3700, 3420, 2960, 2850, 2360, 1400 cm¹; 1H NMR(CDCl3, 300 MHz) 7.815 (d, J=7.8 Hz, 1H), 7.647 (t, J=7.5 Hz, 1H), 7.493(d, J=6.6 Hz, 2H), 7.317 (d, J=8.1 Hz, 1H), 7.207 (t, J=7.2 Hz, 1H),7.003 (d, J=8.7 Hz, 1H), 6.869 (s, 1H); 13C NMR (CDCl3, 75 MHz) 184.577,165.84, 147.89, 146.85, 145.76, 136.61, 126.71, 124.95, 124.70, 123.44,122.02, 115.07, 114.00, 113.42, 112.93, 56.09.

5. (Z)-2-(2-hydroxy-3-methoxybenzylidene)benzofuran-3(2H)-one (9055)

The crude solid was purified by washing with diethyl ether to yield 47.5mg (17.69%) of 9055 as a solid (MP=Decomp. 211° C.). IR (neat, thinfilm): 3300 (br), 1710, 1650, 1600, 1500, 1390, 1110, 750 cm⁻¹; ¹H NMR(CDCl₃, 300 MHz): 7.90 (dd, J_(1,3)=7.8 Hz, J_(1,2)=1.5 Hz, 1H), 7.82(dd, J_(1,3=7.5) Hz, J_(1,2)=1.3 Hz, 1H), 7.64 (ddd, J=8.4 Hz, J=7.2 Hz,J=0.9 Hz, 1H), 7.47 (s, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.21 (t, J=7.5 Hz,1H), 6.96 (t, J=7.8 Hz, 1H), 6.91, (dd, J_(1,3)=8.1 Hz, J_(1,2=1.5) Hz,1H), 6.26 (s, 1H), 3.93 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz): 184.718,166.060, 147.069, 146.605, 146.241, 136.715, 124.738, 123.561, 123.383,121.967, 119.891, 118.966, 112.997, 111.985, 107.031, 56.238.

6. (Z)-2-(2-bromo-4,5-dimethoxybenzylidene)benzofuran-3(2H)-one (9056)

The crude solid was purified by washing with diethyl ether to yield 9.8mg (4.29%) of 9056 as a yellow solid (MP=144-147° C.). IR (neat, thinfilm): 2980, 1700, 1550, 1500, 1100 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): 7.91(s, 1H), 7.79 (d, J=7.0 Hz, 1H), 7.63 (ddd, J=8.4 Hz, J=7.2 Hz, J=0.9Hz, 1H), 7.26 (d, J=8.0 Hz, 1H), 7.24 (s, 1H), 7.21 (t, J=7.5 Hz, 1H),7.08 (s, 1H), 3.97 (s, 3H), 3.91 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz):184.331, 165.772, 150.880, 148.401, 146.560, 136.811, 124.838, 124.419,123.694, 121.771, 118.871, 115.754, 114.084, 112.872, 111.470, 56.309,56.147.

7. (Z)-2-((5-bromothiophen-2-yl)methylene)benzofuran-3(2H)-one (9058)

The crude solid was purified by washing with diethyl ether to yield110.8 mg (36.07%) of 9058 as a yellow solid (MP=155-158° C.). IR (neat,thin film): 3100, 1700, 1640, 1600, 1410, 1120, 890, 760 cm⁻¹; ¹H NMR(CDCl₃, 300 MHz): 7.79 (dd, J_(1,3)=7.5 Hz, J_(1,2)=0.6 Hz, 1H), 7.66(t, J=7.8 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 7.32 (m, 2H), 7.10 (d, J=3.9Hz, 1H), 7.05 (s, 1H); ¹³C NMR (CDCl₃, 75 MHz): 183.737, 165.641,145.485, 137.423, 136.929, 133.095, 130.933, 124.712, 123.772, 122.251,119.695, 113.098, 106.296.

8. (Z)-2-((5-methyl-H-imidazol-4-yl)methylene)benzofuran-3(2H)-one(9059)

The crude solid was purified by washing with diethyl ether to yield 42.2mg (18.63%) of 9059 as an orange solid (MP=180-183° C.). IR (neat, thinfilm): 2900, 1710, 1590, 1450, 1390, 750 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz):7.81 (m, 2H), 7.64 (ddd, J=8.6, 7.3, 1.4 Hz, 1H), 7.29 (d, J=8.4 Hz,1H), 7.24 (t, J=7.5 Hz, 1H), 6.97 (s, 1H). ¹³C NMR (CDCl₃, 125 MHz):183.20, 164.87, 144.02, 139.34, 137.79, 136.58, 124.72, 123.75, 123.26,119.81, 113.43, 90.01, 12.14

9. (Z)-2-((3-bromothiophen-2-yl)methylene)benzofuran-3(2H)-one) (9060)

The crude solid was purified by washing with diethyl ether to yield163.1 mg (53.11%) of 9060 as an orange solid (MP=176-180° C.). IR (neat,thin film): 3100, 3030, 1710, 1650, 1600, 1300, 1180, 1100, 870, 780,720 cm¹; ¹H NMR (CDCl₃, 300 MHz) 7.81 (ddd, J=7.4, 1.2, 0.5 Hz, 1H),7.66 (ddd, J=8.7, 7.3, 1.4 Hz, 1H), 7.59 (dd, J=5.3, 0.9 Hz, 1H), 7.34(d, J=8.3 Hz, 1H), 7.31 (d, J=0.9 Hz, 1H), 7.23 (t, J=7.5 Hz, 1H), 7.12(d, J=5.4 Hz, 1H); ¹³C NMR (CDCl₃, 75 MHz): 183.72, 165.63, 146.08,136.93, 131.44, 130.95, 130.89, 124.76, 123.80, 122.20, 117.67, 113.11,105.23.

10. (Z)-2-((4-bromothiophen-2-yl)methylene)benzofuran-3(2H)-one (9061)

The crude solid was purified by washing with diethyl ether to yield 40.4mg (13.14%) of 9061 as a yellow solid (MP=125-129° C.). IR (neat, thinfilm): 2980, 1700, 1050, 1000 cm⁻¹; H NMR (CDCl₃, 300 MHz): 7.80 (d,J=7.5 Hz, 1H), 7.67 (t, J=7.2 Hz, 1H), 7.46 (s, 2H), 7.34 (dd, J=8.4 Hz,J=0.6 Hz, 1H), 7.24 (t, J=7.5 Hz, 1H), 7.02 (s, 1H); ¹³C NMR (CDCl₃, 75MHz): 183.852, 165.778, 146.059, 137.100, 136.669, 134.353, 128.264,124.774, 123.872, 121.985, 113.121, 111.480, 105.217.

11. (Z)-2-((1H-indol-3-yl)methylene)benzofuran-3(2H)-one (9062)

The crude solid was purified by washing with 50% ethanol to yield 11.2mg (4.29%) of 9062 as an dark red solid (Decomp. at 206-208° C.). IR(neat, thin film): 3000, 2890, 1670, 1590, 1380, 1120, 750 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz): 8.93 (s, 1H), 8.18 (d, J=2.8 Hz, 1H), 7.92 (dd, J=6.1,2.4 Hz, 1H), 7.82 (dd, J=7.6, 1.0 Hz, 1H), 7.62 (d, J=8.5, 7.0, 1.0 Hz,1H), 7.45 (dd, J=6.4, 2.4 Hz, 2H), 7.40 (s, 2H), 7.32 (d, J=8.1 Hz, 2H),7.30-7.27 (m, 3H), 7.20 (t, J=7.5 Hz, 2H).

12. (Z)-2-((1H-indol-4-yl)methylene)benzofuran-3(2H)-one (9063)

The crude solid was purified by washing with diethyl ether to yield112.1 mg (42.92%) of 9063 as an orange solid (MP=230-238° C.). IR (neat,thin film): 2980, 1690, 1640, 1590, 1120, 1060, 900 cm⁻¹; H NMR (CDCl₃,300 MHz): 8.36 (s, 1H), 8.28 (s, 1H), 7.84 (m, 2H), 7.65 (ddd, J=8.6 Hz,J=7.3 Hz, J=1.4 Hz, 1H), 7.47 (d, J=8.5 Hz, 1H), 7.38 (d, J=8.3 Hz, 1H),7.28 (dd, J=3.2 Hz, J=2.5 Hz, 1H), 7.22 (t, J=7.5 Hz, 1H), 7.10 (s, 1H),6.68 (m, 1H). ¹³C NMR (CDCl₃, 75 MHz): 184.75, 165.96, 145.85, 136.78,136.47, 128.53, 126.05, 125.72, 125.48, 124.63, 124.41, 123.23, 122.23,115.89, 113.03, 111.72, 103.89.

13. (Z)-2-(3-methylbenzylidene)benzofuran-3(2H)-one (9064)

The crude solid was purified by washing with 10% ether in hexanes toyield 11.2 mg (xx %) of 9064 as a yellow solid (MP=74-75° C.). IR (neat,thin film): 3040, 2980, 1710, 1610, 1310, 1210, 1150, 900, 750, 700; ¹HNMR ((CDCl₃, 300 MHz) 7.81 (dd, J=7.7, 0.8 Hz, 1H), 7.76 (d, J=7.7 Hz,1H), 7.71 (s, 1H), 7.66 (ddd, J=8.5, 7.3, 1.4 Hz, 1H), 7.35 (m, 2H),7.22 (m, 2H), 6.88 (s, 1H), 2.43 (s, 3H). ¹³C NMR ((CDCl₃, 75 MHz) δ184.95, 166.24, 146.91, 138.65, 136.95, 132.29, 132.25, 130.95, 128.91,128.85, 124.77, 123.53, 121.78, 113.45, 113.07, 21.55.

14. (Z)-2-(4-methylbenzylidene)benzofuran-3(2H)-one (9065)

The crude solid was purified by washing with diethyl ether to yield 34.9mg of 9065 as a tan solid (MP=75-76° C.). IR (neat, thin film): 3020,2920, 1700, 1650, 1600, 1490, 1300, 1200, 1110, 900, 750 cm⁻¹ ¹H NMR(CDCl₃, 300 MHz): 7.8 (m, 3H), 7.64 (ddd, J=8.6, 7.3, 1.4 Hz, 1H), 7.32(d, J=8.3 Hz, 1H), 7.23 (m, 3H), 6.89 (s, 1H), 2.40 (s, 3H); ¹³C NMR(CDCl₃, 75 MHz): 184.88, 166.12, 146.61, 140.63, 136.86, 131.70, 129.81,129.60, 124.71, 123.46, 121.85, 113.53, 113.03, 21.75.

15. (Z)-2-(4-hydroxybenzylidene)benzofuran-3(2H)-one (9068)

The crude solid was purified by washing with diethyl ether to yield 81.7mg (34.29%) of 9068 as a yellow-orange solid (MP=258-260° C.). IR (neat,thin film): 2980, 1700, 1560, 1300, 790 cm⁻¹; ¹H NMR (DMSO-D₆, 300 MHz):10.35 (s, 1H), 7.85 (m, 2H), 7.75 (m, 2H), 7.52 (d, J=8.2 Hz, 1H), 7.27(m, 1H), 6.91 (m, 3H); ¹³C NMR (DMSO-D₆, 75 MHz) 183.67, 165.53, 160.46,145.23, 137.69, 134.28, 124.62, 124.24, 123.31, 121.81, 116.76, 114.03,113.71.

16. (Z)-3-((3-oxobenzofuran-2(3H)-ylidene)methyl)benzonitrile (9070)

The crude solid was purified by washing with diethyl ether to yield 95.3mg (38.54%) of 9070 as a white solid (MP=175-177° C.). IR (neat, thinfilm): 3020, 2200, 1700, 1650, 1490, 1300, 750 cm⁻¹; ¹H NMR (CDCl₃, 300MHz) 8.31 (t, J=1.6 Hz, 1H), 8.02 (dt, J=7.9, 1.4 Hz, 1H), 7.82 (dd,J=7.6, 0.9 Hz, 1H), 7.69 (m, 2H), 7.56 (t, J=7.8 Hz, 1H), 7.39 (d, J=8.3Hz, 1H), 7.27 (t, J=7.2 Hz, 1H), 6.81 (s, 1H); ¹³C NMR (CDCl₃, 125 MHz):184.56, 166.29, 147.81, 137.60, 135.35, 134.34, 133.64, 132.59, 129.78,124.96, 124.09, 121.22, 118.50, 113.38, 113.14, 109.73.

17. (Z)-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-one (9078)

The crude solid was purified by washing with diethyl ether to yield 45.2mg (16.85%) of 9078 as a brown solid (MP=186-187° C.). IR (neat, thinfilm): 3200 (br), 2920, 1700, 1650, 1600, 1290, 1120, 750 cm⁻¹: ¹H NMR(CDCl₃, 300 MHz): 7.80 (d, J=4.8 Hz, 1H), 7.68 (d, J=1.2 Hz, 1H), 7.64(t, J=4.5 Hz, 1H), 7.36 (dd, J_(1,3)=5.1 Hz, J_(1,2)=1.2 Hz, 1H), 7.33(d, J=4.8 Hz, 1H), 7.20 (t, J=5.4 Hz, 1H), 6.91 (d, J=4.8 Hz, 1H), 6.84(s, 1H); ¹³C NMR (CDCl₃, 75 MHz): 184.75, 166.02, 148.38, 146.17,145.82, 136.74, 125.98, 125.48, 124.65, 123.39, 121.94, 116.96, 113.58,113.05, 110.73, 56.09.

18. (Z)-2-(pyridin-3-ylmethylene)benzofuran-3(2H)-one (9253)

The crude solid was purified by via the use of the aldehyde scavengerp-toluenesulfonyl hydrazine bound to polystyrene to yield 45.3 mg(20.3%) of aurone 9253 as a brown solid (MP=122-123° C.). IR (neat, thinfilm): 3400, 3080, 2100, 1700, 1650, 1600, 1450, 1300, 1180, 1100, 880,775, 750 cm⁻¹, ¹H NMR (CDCl₃, 500 MHz): 9.01 (s, 1H), 8.57 (d, J=4.2 Hz,1H), 8.25 (ddd, J=8.7, 5.2, 1.7 Hz, 1H), 7.77 (ddd, J=17.2, 8.8, 3.8 Hz,1H), 7.65 (m, 1H), 7.37 (dd, J=8.0, 4.8 Hz, 1H), 7.30 (m, 1H), 7.22 (t,J=7.5 Hz, 1H), 6.81 (s, 1H). ¹³C NMR (CDCl₃, 125 MHZ): 184.43, 166.25,152.33, 150.18, 148.10, 137.76, 137.39, 128.65, 124.93, 123.93, 123.84,121.40, 113.06, 108.94.

19. (Z)-2-(ferrocenyl)benzofuran-3(2H)-one (3001)

The crude solid was purified via trituration with diethyl ether to yield113.9 mg (69%) of 3001 as a dark purple solid (MP=162-165) IR (neat,thin film): 1700, 1650, 1600, 1470, 1300, 1200, 1125, 1070, 1000, 950,900, 800, 750, 700 cm⁻¹. ¹H NMR (CDCl₃ 300 MHz): δ 7.81 (dd, J=7.6, 1.0Hz, 1H), 7.64 (t, J=8.4 Hz, 1H), 7.29 (m, 1H), 7.19 (td, J=7.5, 1.6 Hz,1H), 6.90 (s, 1H), 4.87 (s, 2H), 4.55 (s, 2H), 4.18 (s, 5H). ¹³C NMR(CDCl₃ 75 MHz): 183.21, 165.51, 146.13, 136.21, 124.56, 123.16, 122.65,116.59, 113.03, 75.14, 71.91, 71.58, 70.04.

20. (Z)-2-(benzofuran-2-ylmethylene)benzofuran-3(2H)-one (3002)

The crude solid was purified via trituration with diethyl ether to yield91.7 mg (70%) of 3002 as a yellow solid (MP=145-150) IR (neat, thinfilm): 1700, 1650, 1600, 1475, 1300, 1200, 1100, 980, 875, 800, 725cm⁻¹: ¹H NMR (CDCl₃, 300 MHz) 7.81 (ddd, J=7.0, 1.4, 0.7 Hz, 1H), 7.68(m, 2H), 7.55 (d, J=8.3 Hz, 1H), 7.48 (s, 1H), 7.38 (m, 2H), 7.26 (m,3H), 6.98 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz) 183.95, 166.02, 155.82,150.32, 146.99, 137.11, 128.91, 126.51, 124.82, 123.87, 123.60, 121.91,121.88, 113.15, 113.13, 111.70, 101.61. This reaction run at 0.5 mmolscale.

21. (Z)-2-(2,5-dibromobenzylidene)benzofuran-3(2H)-one (3003)

The crude solid was purified via trituration with diethyl ether to yield104.5 mg (55%) of 3003 as a cream white solid (MP=181-182) IR (neat,thin film): 3100, 1700, 1650, 1600, 1450 1400, 1350, 1300, 1200, 1100,1010, 950, 900, 800, 750, 700 cm⁻¹. ¹H NMR (301 MHz, CHLOROFORM-D) δ8.44 (d, J=2.4 Hz, 1H), 7.83 (ddd, J=7.6, 1.4, 0.6 Hz, 1H), 7.69 (ddd,J=8.3, 7.3, 1.4 Hz, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.35 (m, 2H), 7.26 (m,1H), 7.20 (s, 1H). ¹³C NMR (76 MHz, CHLOROFORM-D) δ 184.51, 166.32,147.98, 137.47, 134.78, 134.66, 134.00, 133.57, 125.07, 125.04, 124.08,121.61, 121.35, 113.23, 109.21.

22. (Z)-2-(thiophen-3-ylmethylene)benzofuran-3(2H)-one (3004)

The crude solid was purified via trituration with diethyl ether to yield84.4 mg (74%) of 3004 as a greenish yellow solid (MP=125-126). IR (neat,thin film): 3075, 1700, 1650, 1600, 1450, 1410, 1325, 1300, 1200, 1125,1100, 950, 890, 875, 750, 500 cm⁻¹: ¹H NMR (CDCl₃, 300 MHz) δ 7.80 (ddd,J=7.7, 1.4, 0.6 Hz, 1H), 7.54 (m, 2H), 7.55 (ddd, J=3.7, 1.1, 0.6 Hz,1H), 7.35 (dt, J=8.3, 0.7 Hz, 1H), 7.22 (td, J=7.7, 0.8 Hz, 1H), 7.18(s, 1H), 7.15 (dd, J=5.1, 3.7 Hz, 1H). ¹³C NMR (CDCl₃, 75 MHz) δ 184.01,165.74, 145.42, 136.78, 135.65, 133.23, 131.87, 128.17, 124.66, 123.60,122.34, 113.13, 107.16.

23. (Z)-2-(furan-3-ylmethylene)benzofuran-3(2H)-one (3005)

The reaction yielded pure product in the amount of 90.1 mg (85%) of 3005as a pale yellow solid (MP=111-112). IR (neat, thin film): 3150, 3075,1700, 1650, 1600, 1450, 1300, 1200, 1150, 1120, 1100, 1025, 1000, 925,900, 800, 750, 725, 700. ¹H NMR (CDCl₃, 300 MHz) 7.97 (dd, J=1.4, 0.7Hz, 1H), 7.79 (ddd, J=7.7, 1.4, 0.6 Hz, 1H), 7.64 (ddd, J=8.7, 7.3, 1.4Hz, 1H), 7.55-7.47 (m, 1H), 7.29 (dt, J=8.3, 0.6 Hz, 1H), 7.21 (t, J=7.5Hz, OH), 6.93 (d, J=1.9 Hz, 1H), 6.85 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)184.14, 147.06, 146.28, 144.17, 136.88, 124.69, 123.46, 122.20, 118.97,112.96, 111.09, 104.51.

24. (Z)-N-(4-((3-oxobenzofuran-2(3H)-ylidene)methyl)phenyl)acetamide(3006)

The crude solid was purified via trituration with diethyl ether for toyield 43.3 mg (31% yield) of 3006 as a yellow solid (MP=260-270) IR(neat, thin film): ¹H NMR (301 MHz, CHLOROFORM-D) δ 7.91 (d, J=8.7 Hz,1H), 7.81 (dd, J=7.7, 1.4 Hz, 1H), 7.70-7.59 (m, 2H), 7.34 (d, J=8.4 Hz,1H), 7.25-7.19 (m, 1H), 6.87 (s, 1H), 2.22 (s, 3H).

25. (Z)-2-(4-(tert-butyl)benzylidene)benzofuran-3(2H)-one (3008)

The crude solid was purified via trituration with 25% diethylether/hexanes to yield 45.9 mg (33%) of 3008 as a pale yellow solid(MP=87-90) IR (neat, thin film): ¹H NMR (CDCl₃, 300 MHz) 7.88 (d, J=7.3Hz, 2H), 7.81 (d, J=7.6 Hz, 1H), 7.65 (t, J=7.8 Hz, 1H), 7.49 (d, J=7.2Hz, 2H), 7.33 (d, J=8.3 Hz, 1H), 7.22 (m, 1H), 6.91 (s, 1H), 1.36 (s,9H). ¹³C NMR (76 MHz, CHLOROFORM-D) δ 184.90, 166.15, 153.66, 146.76,136.87, 131.55, 129.60, 126.06, 124.73, 123.47, 121.88, 113.37, 113.03,35.08, 31.24.

26. (Z)-2-(4-butylbenzylidene)benzofuran-3(2H)-one (3009)

The crude solid was purified via trituration with pentane to yield 51.4mg (37%) of 3009 as a light yellow solid (MP=80-81) IR (neat, thinfilm): ¹H NMR (CDCl₃, 300 MHz) 7.83 (m, 3H), 7.65 (ddt, J=8.7, 7.3, 1.4Hz, 1H), 7.34 (dd, J=8.3, 0.7 Hz, 1H), 7.28 (m, 2H), 7.22 (t, J=7.5 Hz,1H), 6.91 (s, 1H), 2.66 (t, J=7.7 Hz, 2H), 1.6 (m, 2H), 1.35 (sex, 2H),0.94 (t, J=8.0, 3H). ¹³C NMR (CDCl₃, 75 MHz) 184.90, 166.15, 153.66,146.76, 136.87, 131.55, 129.60, 126.06, 124.73, 123.47, 121.88, 113.37,113.03, 35.08, 31.24.

27. (Z)-2-((5-bromopyridin-2-yl)methylene)benzofuran-3(2H)-one (3011)

The crude solid was purified via trituration with 50% diethylether/hexanes to yield 120.4 mg (80%) of 3011 as a pale yellow solid(MP=192-193). IR (neat, thin film): 3050, 1700, 1650, 1600, 1550, 14501400 1350, 1200, 1125, 1090, 1025, 925, 900, 830, 800, 700. ¹H NMR(CDCl₃, 300 MHz) 8.77 (s, 1H), 8.14 (dd, J=8.4, 1.6 Hz, 1H), 7.69 (ddd,J=8.3, 7.4, 0.9 Hz, 2H), 7.58 (d, J=8.4 Hz, 1H), 7.32 (d, J=8.3 Hz, 1H),7.26 (t, J=8.3, 7.6 Hz, 2H), 6.77 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)184.30, 166.21, 152.37, 148.37, 142.86, 139.74, 137.60, 128.49, 127.96,125.05, 124.16, 121.31, 113.10, 107.47.

28. (Z)-2-(3,5-dibromo-2-hydroxybenzylidene)benzofuran-3(2H)-one (3012)

The crude solid was purified via trituration with diethyl ether/hexanesto yield 27.7 mg (14%) of 3012 as a yellow solid (MP=decomp 225) IR(neat, thin film): 1H NMR (301 MHz, CHLOROFORM-D) 8.38-8.29 (m, 1H),7.80 (t, J=7.6 Hz, 1H), 7.72-7.61 (m, 2H), 7.37 (d, J=8.5 Hz, 1H),7.33-7.16 (m, 4H), 6.21 (s, 1H).

29. (Z)-2-((6-bromopyridin-2-yl)methylene)benzofuran-3(2H)-one (9260)

1H NMR (500 MHz, CHLOROFORM-D) δ 8.18 (d, J=7.9 Hz, 1H), 7.81 (d, J=8.5Hz, 1H), 7.71-7.62 (m, 2H), 7.45 (d, J=7.9 Hz, 1H), 7.33 (d, J=8.5 Hz,1H), 7.28-7.22 (m, 1H), 6.98 (s, 1H). 13C NMR (126 MHz, CHLOROFORM-D) δ184.60, 166.49, 153.09, 148.69, 142.21, 138.77, 137.53, 127.79, 125.24,125.07, 124.16, 121.27, 113.08, 110.97.

30. (Z)-5-fluoro-2-(pyridin-2-ylmethylene)benzofuran-3(2H)-imine (9312)

This compound is made using the Method #3 (Varma).

Method #3—Methanol/KOH Microwave (Lee et al., Eur. J. Med. Chem., 2010,45 2957-2971; Carrasco et al., Eur. J. Med Chem., 2014, 80:523-534)

In the following syntheses, coumaranone (1.00 mmol) and aldehyde (1.00mmol) were combined in a microwave tube and 5 mL of methanol was thenadded. In a separate vial 0.75 g of KOH and 0.75 g of water werecombined. The KOH solution was then added to the microwave tube. Thetube was then microwaved at 110° C. for 12 minutes. The reaction mixturewas then allowed to cool to room temperature. Once at room temperature,the vial was then washed with EtOAc. 10% HCl was used to neutralize theKOH solution. The solution was then partitioned between EtOAc and water.The EtOAc layer was concentrated to dryness in vacuo to afford thedesired aurone. Further purification was performed as noted.

1. (Z)-2-(4-bromobenzylidene)-6,7-dihydroxybenzofuran-3(2H)-one (1005)

The crude solid was purified by washing with diethyl ether to yield 10.4mg (3.12%) of 1005 as brown/yellow solid (Decomp at 290° C.). IR (neat,thin film): 3200-3600, 2390, 1550, 1100 cm⁻¹; ¹H NMR (DMSO-D₆, 500 MHz):δ 10.78 (s, 1H), 9.69 (s, 1H), 7.95 (d, J=8.6 Hz, 2H), 7.68 (m, 2H),7.12 (d, J=8.4 Hz, 1H), 6.75 (s, 1H), 6.72 (d, J=8.1 Hz, 1H);

2.(Z)-6,7-dihydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-one(1009)

The crude solid was purified by washing with diethyl ether to yield 30.1mg (10.02%) of 1009 as a yellow-brown solid (MP=275-280° C.). IR (neat,thin film): 3100-3500, 1700, 1400, 1200 cm⁻¹; ¹H NMR (DMSO, 300 MHz)10.78 (s, 1H), 9.27 (s, 1H), 7.53 (d, J=2.1 Hz, 1H), 7.42 (dd,J_(1,3)=8.4 Hz, J_(1,2)=2.1 Hz, 1H), 7.09 (d, J=8.1 Hz, 1H), 7.01 (d,J=8.4 Hz, 1H), 6.75 (d, J=8.4 Hz, 1H), 6.62 (s, 1H), 3.81 (s, 3H); ¹³CNMR (DMSO, 75 MHz) 182.637, 155.589, 154.924, 150.004, 147.090, 146.835,130.683, 125.418, 124.729, 118.283, 115.803, 114.911, 113.296, 112.622,111.741.

3. (Z)-6-hydroxy-2-(4-methylbenzylidene)benzofuran-3(2H)-one (2904)

The product formed an emulsion which provided a yield of 117.7 mg(46.71%) of 2904 as a yellow solid (MP=258-264° C.). IR (neat, thinfilm): 3091, 1672, 1637, 1575 cm⁻¹; ¹H NMR (DMSO, 500 MHz) 11.29 (s,1H), 7.83 (d, J=8.05 Hz, 2H), 7.60 (d, J=8.6 Hz, 1H), 7.29 (d, J=8.05Hz, 2H), 6.82 (d, J=1.7 Hz, 1H), 6.75 (s, 1H), 6.70 (dd, J=8.4 Hz, J=1.9Hz, 1H), 2.32 (s, 3H); ¹³C NMR (DMSO, 125 MHz) 181.9628, 168.3707,167.1117, 147.4818, 140.2756, 131.6386 ppm, 130.1983 ppm, 129.8263,126.4498, 113.6017, 113.3441, 111.1074, 99.1606, 21.6808.

4. (Z)-2-(4-bromobenzylidene)-6-hydroxybenzofuran-3(2H)-one (2905)

The crude solid was purified by washing with diethyl ether to yield119.9 mg (37.82/%) of 2905 as a yellow solid (MP=170-178° C.). IR (neat,thin film): 3496.4, 3383.8, 2901.7, 1673.9, 1639.5, 1594.2 cm⁻¹; ¹H NMR(DMSO, 500 MHz) 11.472 (s, 1H), 7.880 (d, J=8.55 Hz, 2H) 7.680 (d, J=8.6Hz, 2H), 7.615 (d, J=8.55 Hz, 1H), 6.852 (d, J=1.7 Hz, 1H), 6.776 (s,1H), 6.740 (d, J=10.3, 1H); C NMR (DMSO-D₆, 125 MHz) 181.91, 168.47,167.47, 148.25, 133.36, 132.55, 131.93, 126.58, 123.59, 113.82, 113.05,109.54, 99.21.

5. (Z)-2-((1H-pyrrol-2-yl)methylene)-6-hydroxybenzofuran-3(2H)-one(2906)

The crude solid was purified by washing with diethyl ether to yield212.0 mg (93.40%) of 2906 as a black solid (Decomp 205° C.). IR (neat,thin film): 3130.3, 2360.5, 1622.3, 1597.4 cm⁻¹; ¹H NMR (Acetone-D₆, 300MHz): 10.75 (s, 1H), 7.59 (d, J=8.4 Hz, 1H), (7.65 Minor isomer, d,J=8.4 Hz, 0.33H), 7.14 (td, J=2.7, 1.4 Hz, 1H), (7.23 Minor isomer, dd,J=2.3, 1.3 Hz, 0.33H), 6.73 (m, 4H), 6.30 (m, 1H). ¹³C NMR (Acetone D₆,75 MHz) 180.772, 167.598, 167.426, 166.1730, 145.666, 145.293, 127.032,126.334, 126.085, 115.616, 114.249, 113.341, 104.373, 104.201, 99.335,98.656.

6. (Z)-6-hydroxy-2-(4-(trifluoromethyl)benzylidene)benzofuran-3(2H)-one(2909)

Reaction yielded pure product in the amount of 194.0 mg (63.35%) ofaurone 2909 as a brown solid (MP=168-174° C.). IR (neat, thin film):3484.3, 3070.8, 1689.5, 1647.3, 1594.5 cm⁻¹; ¹H NMR (Acetone, 300 MHz)8.190 (d, J=8.22 Hz, 2H), 7.830 (d, J=7.92 Hz, 2H), 7.640 (d, J=8.58 Hz,1H), 6.861 (d, J=1.71 Hz, 1H), 6.815 (d, J=10.65 Hz, 1H), 6.766 (s, 1H);¹³C NMR (Acetone D₆, 75 MHz) 181.69, 168.73, 166.52, 149.06, 136.66,131.44, 130.24, 126.15 (q, J=270 Hz), 126.11, 125.67, 113.45, 108.01,98.84

7. (Z)-6-hydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-one(2911)

The crude solid was purified by washing with MeCl₂ to yield 230.0 mg(80.91%) of 2911 as a brown solid (MP=145-151° C.). IR (neat, thinfilm): 1625, 1456.7, 1280, 1120 cm⁻¹; ¹H NMR (Acetone D₆, 300 MHz) 7.575(d, J=0.0275 Hz, 1H), 7.468 (d, J=1.71 Hz, 1H), 7.317 (d, J=8.22 Hz,1H), 6.990 (d, J=8.58 Hz, 1H), 6.776 (d, J=1.71 Hz, 1H), 6.690 (d,J=8.58 Hz, 1H), 6.633 (s, 1H), 3.795 (s, 3H); ¹³C NMR (Acetone D₆, 75MHz) 181.7530, 168.0717, 166.8862, 150.0882, 147.1339, 146.5986,126.3396, 125.2688, 125.2635 (Two overlapping carbons), 117.8306,113.5379, 112.6774, 111.8839, 98.9579, 56.1167.

Method #4—Methanol/Room Temperature (Lee et al., Eur. J. Med. Chem. 452957-2971)

In the following synthesis, coumaranone (1.00 mmol) and aldehyde (1.00mmol) were combined in a vial and 5 mL of methanol was then added. In aseparate vial 0.75 g of KOH and 0.75 g of water were combined. The KOHsolution was then added to vial. The reaction mixture was stirred for 3hours at room temperature. 10% HCl was used to neutralize the KOHsolution. The solution was then partitioned between EtOAc and water. TheEtOAc layer was concentrated to dryness in vacuo to afford the desiredaurone. Further purification was performed as noted.

1.(Z)-4-((4,6-dimethoxy-3-oxobenzofuran-2(3H)-ylidene)methyl)benonitrile(7002)

The crude solid was purified by washing with ethyl acetate to yield 45.0mg (14.64%) of 7002 as a tan solid (MP=220-223° C.). IR (neat, thinfilm): 2359, 2225, 1697, 1617, 1592, 1507, 1473, 1428, 1363, 1253, 1215,1160, 1091, 1053, 1011 cm⁻¹; ¹H NMR (DMSO D₆, 300 MHz): 7.92 (d, J=8.58Hz, 2H), 7.68 (d, J=8.58 Hz, 2H), 6.69 (s, 1H), 6.34 (S, 1H), 6.15 (s,1H), 3.91 (s, 3H), 3.92 (s, 3H).

2. (Z)-2-((3-oxobenzofuran-2(3H)-ylidene)methyl)benzonitrile (3007)

Product precipitated out of solution upon addition of acid to yield101.3 mg (82% yield) of 3007 as a pale yellow solid (MP=decomp 215) IR(neat, thin film): ¹H NMR (301 MHz, ACETONE-D6) δ 7.95 (dd, J=7.7, 1.4Hz, 1H), 7.73 (dd, J=8.0, 1.6 Hz, 1H), 7.66 (dd, J=7.4, 1.5 Hz, 1H),7.61 (dd, J=7.5, 1.6 Hz, 1H), 7.55 (dd, J=7.3, 1.6 Hz, 1H), 7.33 (ddd,J=8.7, 7.2, 1.6 Hz, 1H), 6.83 (dd, J=8.3, 1.0 Hz, 1H), 6.77 (t, J=7.6Hz, 1H), 5.88 (s, 1H).

Method #5—Ethanol/KOH Microwave (Lee et al., Eur. J. Med. Chem. 452957-2971)

In the following synthesis, the same procedure as Method #3 wasemployed, but using ethanol in place of methanol.

1. (Z)-6-hydroxy-2-(2,3,4-trimethoxybenzylidene)benzofuran-3(2H)-one(2912)

Reaction yielded pure product in the amount of 259.0 mg (78.89%) of 2912as a reddish brown solid (MP=225-230° C.). IR (neat, thin film): 3444.7,1585.0, 1277.2, 1140.3, 1101.5 cm⁻¹; ¹H NMR (Acetone-D₆, 300 MHz) 8.015(d, J=8.58 Hz, 1H), 7.610 (d, J=8.25 Hz, 1H), 7.036 (s, 1H), 6.935 (d,J=8.91 Hz, 1H), 6.810 (d, J=2.07 Hz, 1H), 6.775 (d, J=8.22 Hz, 1H) 3.939(s, 3H), 3.916 (s, 3H), 3.819 (s, 3H); ¹³C NMR (Acetone-D₆, 75 MHz)181.580, 168.062, 165.595, 155.585, 153.654, 147.334, 142.238, 126.607,125.737, 119.063, 114.140, 112.562, 108.174, 104.311, 98.508, 61.279,60.461, 55.485.

Method #6—AcOH/HCl (Cheng et al., Eur. J. Med. Chem. 2010, 45,5950-5957)

In the following synthesis, coumaranone (1.00 mmol) and aldehyde (1.00mmol) were combined in a flask with 10 mL glacial acetic acid and 4drops concentrated HCl. It was allowed to stir at room temperature for 3hours. It was then worked-up using ice water. A precipitate was formedand filtered off.

1. (Z)-4-((6-methoxy-3-oxobenzofuran-2(3H)-ylidene)methyl)benzonitrile(5006)

The crude solid was purified by washing with diethyl ether to yield 179mg (65%) of 5006 as a brown solid (MP=180-181° C.). IR (neat, thinfilm): 3330, 2210, 1720, 1600, 1450, 1280, 1040, 920, 840, 760, 660; ¹HNMR (Acetone D₆, 300 MHz): 7.98 (d, 7.89 Hz, 2H), 7.74 (d, 3.15 Hz, 1H),7.73 (s, 1H), 7.71 (d, 2.4 Hz, 1H), 6.78 (d, 6.87 Hz, 2H), 6.76 (s, 1H),4.00 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) 182.75, 168.85, 168.01, 149.40,137.06, 132.53, 131.42, 126.28, 118.79, 114.42, 112.71, 112.40, 109.02,96.94, 56.26.

Method #7—Handy Microwave (Taylor et al., Tetrahedron Lett., 2017,58(3):240-241)

In the following syntheses, coumaranone (1.00 mmol) and aldehyde (1.00mmol) were combined in a microwave vial. 1 mL of the deep eutecticsolvent formed from a 1:2 molar ratio of choline chloride and urea wasadded. The tube was then microwaved at 90° C. for 30 minutes. At thispoint, the reaction was allowed to cool to room temperature andpartitioned between water and methylene chloride. The organic layer wasseparated and concentrated to dryness in vacuo to afford the desiredaurone. Further purification was performed as noted.

1. (Z)-2-(4-methoxybenzylidene)benzofuran-3(2H)-one (6601)

The crude solid was purified by washing with diethyl ether to yield 38.9mg (15.4%) of 6601 as a red-orange solid (MP=135-138° C.). IR (neat,thin film): 3020, 3000, 1700, 1670, 1600, 1510, 1240, 900, 820, 750cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) 7.90 (d, J=8.91 Hz, 2H), 7.80 (d, J=6.87Hz, 1H), 7.65 (t, J=7.2 Hz. 1H), 7.31 (d, J=8.25 Hz, 1H), 7.21 (t, J=7.2Hz, 1H), 7.00 (d, J=8.94 Hz, 2H), 6.89 (s, 1H), 3.87 (s, 3H); ¹³C NMR(CDCl₃, 75 MHz): 184.67, 165.92, 161.16, 145.97, 136.64, 133.55, 125.14,124.65, 123.37, 122.03, 114.59, 113.52, 112.97, 55.49.

2. (Z)-2-benzylidenebenzofuran-3(2H)-one (6615)

The crude solid was purified by washing with diethyl ether to yield 32.7mg (14.70%) of 6615 as a yellow solid (MP=92-96° C.). IR (neat, thinfilm): 3050, 3010, 1700, 1650, 1600, 1480, 1290, 1120, 890, 740, 690cm⁻¹; ¹HNMR (CDCl₃, 500 MHz) 7.94 (d, J=8.60 Hz, 2H), 7.81 (d, J=7.45Hz, 1H), 7.66 (t, J=8.6 Hz, 1H), 7.47 (t, J=7.45 Hz, 2H), 7.41 (d,J=7.40 Hz, 1H), 7.34 (d, J=8.6 Hz, 1H), 7.23 (t, J=7.45 Hz, 1H), 6.91(s, 1H) ¹³CNMR (CDCl₃, 75 MHz): 184.71, 166.39, 146.86, 137.04, 132.39,131.66, 130.02, 129.01, 124.80, 123.59, 121.73, 113.20, 113.06.

3.(2Z,2′Z)-2,2′-(1,4-phenylenebis(methanylylidene))bis(benzofuran-3(2H)-one)(6617)

The reaction yielded 121.7 mg (33.3%) of 6617 as an orange solid (Decomp189° C.). IR (neat, thin film): 3030, 1710, 1600, 1470, 1290, 1180,1100, 900, 750 cm⁻¹; ¹HNMR (CDCl₃, 500 MHz) 8.01 (s, 3H), 7.82 (d.J=8.05 Hz, 2H), 7.69 (ddd, J=1.7, 7.45, 8.6 Hz, 4H), 7.36 (d, J=8.05 Hz,3H), 6.92 (s, 2H); ¹³CNMR (CDCl₃, 125 MHz) 184.87, 166.22, 147.57,137.21, 133.66, 131.96, 124.90, 123.82, 121.67, 113.04, 112.11.

4. (Z)-2-(2-methylbenzylidene)benzofuran-3(2H)-one (9057)

The crude solid was purified by washing with 50:50 diethyl ether/Hexanesto yield 61.9 mg (26.20%) of 9057 as a yellow solid (MP=89-93° C.). IR(neat, thin film): 3030, 2920, 1710, 1650, 1600, 1300, 1100, 750 cm⁻¹;¹H NMR (CDCl₃, 300 MHz): 8.26 (d, J=8.6 Hz, 1H), 7.82 (d, J=7.6 Hz, 1H),7.65 (t, J=8.2 Hz, 1H), 7.29 (m, 5H), 7.14 (s, 1H), 2.52 (s, 3H)¹³C NMR(CDCl₃, 75 MHz): 184.92, 166.32, 147.08, 139.33, 136.98, 131.28, 130.89,130.80, 129.96, 126.50, 124.82, 123.53, 121.81, 113.08, 110.02, 20.15.

5. (Z)-2-((5-(hydroxymethyl)furan-2-yl)methylene)benzofuran-3(2H)-one(9067)

The crude reaction mixture was then purified by trituration with etherto afford 48.9 mg (20%) of 9067 as a black solid (MP=61-63° C.). IR(neat, thin film): 3360, 2920, 1689, 1639, 1598, 1479, 1402, 1300 cm⁻¹;1H NMR (CDCl₃, 300 MHz) 7.76 (d, J=6.8 Hz, 1H), 7.70 (t, J=6.8 Hz, 1H),7.34 (d, J=6.8 Hz, 1H), 7.22 (J=6.8 Hz, 1H), 7.11 (d, J=4.5 Hz, 1H),6.88 (s, 1H), 6.53 (d, J=4.5 Hz, 1H), 5.74 (s, 2H); 13C NMR (CDCl₃, 125MHz) 184.03, 165.66, 157.51, 148.59, 145.10, 136.80, 124.59, 123.59,122.03, 118.48, 112.97, 111.24, 101.81, 57.62.

6. (Z)-4-((5-methyl-3-oxobenzofuran-2(3H)-ylidene)methyl)benzonitrile(4001)

The crude reaction mixture was then purified by trituration with 50%diethyl ether/hexanes to afford 20.1 mg of 4001 as a yellow-brown solid(MP=148-152° C.). IR (neat, thin film): ¹H NMR (CDCl₃, 300 MHz): 7.99(d, J=8.4 Hz, 2H), 7.72 (d, J=8.4 Hz, 2H), 7.60 (s, 1H), 7.51 (d, J=8.1Hz, 1H), 7.24 (m, 1H), 6.79 (s, 1H), 2.42 (s, 3H); 13C NMR (CDCl₃, 75MHz): 184.80, 164.85, 148.67, 138.74, 136.97, 134.02, 132.52, 131.56,124.63, 121.09, 118.68, 112.65, 112.51, 109.76, 22.74.

7. (Z)-2-(4-bromobenzylidene)-5-methylbenzofuran-3(2H)-one (4002)

The crude reaction mixture was then purified by trituration with 50%diethyl ether/hexanes to afford 50.2 mg of 4002 as a yellow solid(MP=171-175° C.). IR (neat, thin film): ¹H NMR (CDCl₃, 300 MHz): 7.75(d, J=8.6 Hz, 2H), 7.56 (m, 3H), 7.46 (dd, J=8.4, 1.9 Hz, 1H), 7.21 (d,J=8.4 Hz, 1H), 6.76 (s, 1H), 2.40 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz):184.88, 164.65, 147.58, 138.31, 133.53, 132.83, 132.21, 131.39, 124.43,124.27, 121.44, 112.60, 111.36, 20.89.

8. (Z)-2-(2-hydroxy-3-methoxybenzylidene)-5-methylbenzofuran-3(2H)-one(4003)

The crude reaction mixture was then purified by trituration with ethanolto afford 10.0 mg of 4003 as a light-yellow solid (MP=134-137° C.). IR(neat, thin film): ¹H NMR (301 MHz, CHLOROFORM-D) δ 7.88 (dd, J=7.8, 1.7Hz, 1H), 7.59 (m, 1H), 7.43 (m, 2H), 7.20 (d, J=8.4 Hz, 1H), 6.93 (m,2H), 2.40 (s, 3H);

9. (Z)-5-methyl-2-(2,3,4-trimethoxybenzylidene)benzofuran-3(2H)-one(4004)

The crude reaction mixture was then purified by trituration with 50%diethyl ether/hexanes to afford 63.0 mg of 4004 as a dark-orange solid(MP=74-78° C.). IR (neat, thin film): 1H NMR (CDCl₃, 300 MHz) 8.07 (d,J=8.9 Hz, 1H), 7.58 (s, 1H), 7.42 (dd, J=8.5, 1.7 Hz, 1H), 7.26 (d,J=4.2 Hz, 1H), 7.17 (d, J=8.4 Hz, 1H), 6.78 (d, J=9.0 Hz, 1H), 3.95 (s,3H), 3.91 (s, 3H), 3.87 (s, 3H), 2.38 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz):184.80, 164.30, 155.54, 154.12, 147.09, 137.70, 133.04, 127.36, 124.29,121.97, 119.60, 112.45, 107.76, 107.48, 107.27, 61.97, 61.00, 56.16,20.89.

10. (Z)-2-(4-hydroxybenzylidene)-5-methylbenzofuran-3(2H)-one (4005)

The crude reaction mixture was then purified by trituration with diethylether to afford 21.5 mg of 4005 as an orange-yellow solid (MP=228-231°C.). IR (neat, thin film): 1H NMR (ACETONE-D₆, 300 MHz) 7.88 (d, J=8.6Hz, 2H), 7.57 (ddd, J=8.4, 1.9, 0.5 Hz, 1H), 7.53 (m, 1H), 7.35 (d,J=8.4 Hz, 1H), 6.98 (d, J=8.7 Hz, 2H), 6.77 (s, 1H), 2.39 (s, 3H);

11. (Z)-5-methyl-2-(2-(trifluoromethyl)benzylidene)benzofuran-3(2H)-one(4006)

The crude reaction mixture was then purified by trituration with 50%diethyl ether/hexanes to afford 29.6 mg of 4006 as a yellow-brown solid(MP=121-124° C.). IR (neat, thin film): 1H NMR (CDCl₃, 500 MHz): 8.39(d, J=7.9 Hz, 1H), 7.74 (d, J=7.8 Hz, 1H), 7.65 (t, J=7.7 Hz, 1H), 7.59(s, 1H), 7.46 (m, 2H), 7.19 (d, J=8.4 Hz, 1H), 7.15 (d, J=1.5 Hz, 1H),2.40 (s, 3H);

12. (Z)-5-methyl-2-((5-methylfuran-2-yl)methylene)benzofuran-3(2H)-one(9251)

The reaction yielded 138.6 mg (61.31%) of 9251 as a light brown solid(MP=62-64° C.). IR (neat, thin film): 3010, 2850, 1710, 1650, 1590,1520, 1300, 1190, 750 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): 7.78 (d, J=6.7 Hz,1H), 7.62 (ddd, J=8.5, 7, 1 Hz, 1H), 7.30 (d, J=7.9 Hz, 1H), 7.20 (t,J=7.4 Hz, 1H), 7.07 (d, J=3.4 Hz, 1H), 6.86 (s, 1H), 6.22 (d, J=3.2 Hz,1H), 2.41 (s, 3H). ¹³C NMR (CDCl₃, 125 MHz): 183.82, 165.67, 156.61,147.56, 144.32, 136.43, 124.60, 123.21, 122.34, 119.34, 112.81, 110.28,102.22, 14.30.

13. (Z)-2-(3-hydroxybenzylidene)benzofuran-3(2H)-one (9252)

The crude reaction mixture was then purified by trituration with etherto afford 201.2 mg (84.52%) of 9252 as a green-yellow solid(Decomp.=86-89° C.). IR (neat, thin film): 3010, 1700, 1650, 1600, 1450,1120, 890, 750 cm⁻¹; ¹H NMR (DMSO-D₆, 500 MHz): 7.78 (m, 2H), 7.52 (d,J=8.7 Hz, 1H), 7.41 (s, 1H), 7.37 (d, J=7.5 Hz, 1H), 7.28 (m, 2H), 6.83(m, 2H). ¹³C NMR (DMSO-D₆, 75 MHz): 184.19, 165.96, 146.69, 138.26,133.40, 130.50, 124.87, 124.52, 123.11, 123.05, 121.45, 118.23, 118.16,113.71, 113.16.

14. (Z)-2-((1H-imidazol-5-yl)methylene)benzofuran-3(2H)-one (6621)

The reaction yielded 56.2 mg (26.5%) of 6621 as a red solid (MP=° C.).¹H NMR (CDCl₃. 300 MHz): 7.8 (s, 1H), 7.74 (d, 1H), 7.65 (s, 1H), 7.59(m, 1H), 7.22 (d, 1H), 7.18 (t, 1H), 6.97 (s, 1H).

15. (Z)-6-hydroxy-2-(4-methoxybenzylidene)benzofuran-3(2H)-one (6620)

This compound was also synthesized using the Method #7.

Example II. Evaluation of Aurone-Based Compounds for Anti-TrypanosomalActivity Introduction

Neglected tropical diseases constitute a diverse group of diseases thatimpact primarily the poorest populations, affecting more than 1.4billion people worldwide (World Health Organization, Neglected TropicalDiseases, available fromhttp://www.who.int/neglected_diseases/diseases/en/). Several of thesediseases, exemplified by Chagas disease (American trypanosomiasis),human African trypanosomiasis (HAT or African Sleeping Sickness), andthe Leishmaniases (a set of trypanosomal diseases) are caused byparasitic protozoa known as trypanosomatids (commonly referred to astrypanosomes) which are transmitted to human and animal hosts byhematophagous insect vectors. HAT is mainly caused by Trypanosoma bruceiand is transmitted by Tsetse flies; Chagas disease is caused byTrypanosoma cruzi and is transmitted by triatomine bugs; andleishmaniases are caused by various species of Leishmania and aretransmitted by sanctifies.

An estimated 30,000 people are infected and up to 70 million are at riskof developing HAT, which causes severe and progressively fatal centralnervous system impairment. The disease is endemic to the Africancontinent where several subspecies of the parasite T. brucei are spreadby the Tsetse fly. Although HAT is primarily caused by two subspecies ofT. brucei, rhodesiense and gambiense, there have been reports of humaninfections caused by T. evansi, T. lewisi, T. brucei brucei, and T.congolense (WHO fact sheet 2014). Species and subspecies such as T.brucei, T. congolense, T. equiperthem, T suis and T. vivax are known tocause disease (e.g., nagana) in wild animals and domestic animals, suchas cattle.

The choice of treatment during the early stage of HAT depends on thesubspecies of T. brucei responsible for the infection (U. S. Centers forDisease Control and Prevention, African Trypanosomiasis—Resources forHealth Professionals, available fromhttp://www.cdc.gov/parasites/sleepingsickness/health_professionals/index.html).Pentamidine, which was discovered in 1941, is given by intravenousinfusion and is typically used to treat first stage infections caused byT. b. gambiense. The drug can have significant side effects includingleukopenia, thrombopenia, hypotension, arrhythmias, gastrointestinaldistress hepatomaegaly, hepatitis, hypoglycemia, neurological issuesincluding seizures, and nephrotoxicity. Suramin is used to treat firststage HAT when caused by T. b. rhodesiense. Suramin was developed in1916 by Bayer and is also sold under the brand name Germanin. Inaddition to nausea and vomiting, more than 50% of those treated withsuramin will experience adrenal cortical damage which is usuallytemporary.

Treatments for the second stage of HAT are more challenging to developdue to the need to cross the blood-brain barrier to be effective.Melarsoprol (currently produced by Sanofi-Aventis) was discovered to beeffective against late stage HAT in 1949. It is used as a treatment forboth forms of HAT and is the only treatment available for late stageinfections caused by T. b. rhodesiense. Melarsoprol is an arsenicderivative and has significant side effects that are similar to arsenicpoisoning. Due to the dangers of treatment, it is only administered byinjection with close physician supervision. Eflornithine, marketed bySanofi-Aventis as Ornidyl in the United States, is used to treat secondstage disease caused by T. b. gambiense. Although considered somewhatsafer than melarsoprol, the side effects of eflornithine can includeseizures, fever, neutropenia, hypertension and diarrhea. The treatmentregimen, which includes multiple intravenous injections over 14 days, isstrict and very difficult to administer, especially in a rural setting.Strains that are resistant to eflornithine have been reported since the1980s, which has prompted the more recent use of eflornithine incombination with nifurtimox.

Nifurtimox (marketed as Lampit by Bayer) was originally developed fortreatment of Chagas Disease, but has been approved by the WHO fortreatment of HAT. Side effects of nifutimox include gastrointestinal,cardiac and neurological issues and it should not be used by patientswith neurologic or psychiatric disorders. Nifurtimox and eflornithinecombination therapy (NECT) to treat late stage HAT caused by T. b.gambiense was introduced in 2009 (World Health Organization,Trypanosomiasis, human African (sleeping sickness): Media Centre FactSheet, available from:http://www.who.int/mediacentre/factsheets/fs259/en/). The WHO nowprovides NECT at no cost to endemic countries.

Chagas disease affects approximately 6 million people per year and killsapproximately 12,000 people per year. Flu-like symptoms may follow theinitial exposure to the infectious agent, T. cruzi. Most infectedpersons remain asymptomatic for the remainder of their lives; however,approximately 30% of infected persons will progress to the chronic formof Chagas disease after 10-30 years, when cardiac and gastrointestinaldamage usually results in death. T. cruzi is transmitted through thefeces of triatomines, a blood-sucking Reduuvid insect vector, commonlyknown as the Kissing Bug. Reduuvid is endemic in South America and hasalso been found in the southern United. States where Chagas disease hasrecently been classified as an emerging infectious disease threat. Inaddition to the human toll it causes, Chagas disease also affects wildanimals, companion animals and domesticated animals.

Benznidazole, originally marketed as Rochagan and Radanil byHoffman-LaRoche, who later donated rights to the Brazilian government,is used to treat the initial (acute) stage of Chagas disease (U. S.Centers for Disease Control and Prevention, Parasites: AmericanTrypanosomiasis, available from:http://www.cdc.gov/parasites/chagas/epi.html). Although the drug mayprovide some symptomatic relief or slow progression in the chronicstage, there is no known cure for Chagas disease in the chronic stage.Side effects of benznidazole treatment include rash, gastrointestinaldistress, and peripheral neuropathy. Although nifurtimox is still usedas a mainline treatment for Chagas disease, benznidazole is thepreferred treatment due to the serious side effects andcontraindications of nifurtimox.

The leishmaniases encompass three presentations of disease caused byvarious subspecies of Leishmania, which is spread by the bite of thesandfly. More than 12 million people in almost 100 countries worldwideare infected with one of the forms. Cutaneous leishmaniasis (CL) is themost common presentation with an estimated 1.3 million new casesannually. The infection causes ulcerative skin lesions, permanentdisfigurement and serious disability. Mucocutaneous leishmaniasis (ML,also called espundia) causes even more severe disability anddisfigurement due to the destruction of the naso-oropharyngeal mucosa.The most severe form of the disease, visceral leishmaniasis (VL, alsoknown as kala-azar), is caused by various species of Leishmania such asL. donovani and L. major. In this presentation of the disease, thoseinfected exhibit high fever and weight loss, swelling of the spleen andliver, and anemia. The disease is fatal without treatment.Leishmaniasis, like other trypanosomal infections, is also known toaffect wild animals, companion animals and domesticated animals.

Current therapies for Leishmaniasis depend on the form of the disease(U. S. Centers for Disease Control and Prevention.Leishmaniasis—Resources for Health Professionals, 2014, available from:http://www.cdc). Pentavalent antimonials administered by dailyintravenous or intramuscular administration for 10-28 days have beenused to treat all three presentations.

Sodium stibogluconate (manufactured by GlaxoSmithKline as Pentostam®),is the only formation available in the United States, where it has anLND approval from the FDA and is only available through the CDC DrugService. Some of the more serious side effects of treatment includephlebotoxicity, pancreatitis, cardiac conduction abnormalities, andanaphylaxis, Infusion of liposomal atriphotericin B (marketed asAmBisome®) has been approved to treat visceral leishmaniasis (VU since1997. Severe histamine-related reactions have been noted within hours oftreatment. Side effects also include kidney and liver damage,electrolyte imbalance, leukopenia, thrombopenia, and cardiac arrhythmiasand heart failure. The conventional amphotericin B deoxycholate(non-liposomal) is also considered to be very effective for treating VLbut it is much more toxic than the liposomal formulation. Othertreatments for leishmaniasis include the aminoglycoside paromomycinsulfate and pentamidine to treat cutaneous leishmaniasis (CL) andmiltefosine, which was approved by the FDA in 2014 as an oral treatmentfor all three forms of leishmaniasis.

Trypanosomal infection also affects domesticated and companion animals,as well as animals in the wild. Animal African trypanosomiasis (AAT),also known as nagana, dourine and surra, is caused by trypanosomespecies and subspecies other than those affecting human beings.Trypanosoma brucei, Trypanosoma congolese, Trypanosoma equiperdum,Trypanosoma simiae, Trypanosoma suis and Trypanosoma vivax are some ofthe species and subspecies causing diseases in wild and domesticanimals. T. brucei, for example, affects cattle and is a majorimpediment to proper nutrition and economic development in affectedareas of Africa. See, e.g., Seck et al., describing parasitological andserological prevalence data of AAT in Senagal (Parasite, 2010,17(3):257-65). Leishmaniasis also affects cows, horses, dogs, and othercompanion and domesticated animals. Chagas, caused by T. cruzi, is knownto affect dogs and other animals; “rapid tests” are available for canineChagas, but there are no effective treatments available once diagnosed.All these diseases have a serious economic impact on the development ofagriculture in the affected areas. Those affecting cattle areparticularly devastating since they are a major cause for reduced meatand milk production as well as animal power for agricultural production(WHO fact sheet available athttp://www.who.intitrypanosotniasis_african/parasite/en/).

Additionally, animals (as well as humans) serve as disease reservoirs.Typical reservoirs for T. cruzi, for example, include wild animals suchas armadillos, raccoons, opossums, and rodents, as well as domesticatedand companion animals such as dogs, cattle and guinea-pigs (Reza, ChagasDisease, available at http://www.austincc.edu/microbio/2704t/tc). Humansare the main reservoir for Trypanosoma brucei gambiense, but thisprotozoan can also be found in animals. Wild game animals are the mainreservoir of T. b. rhodesiense (Centers for Disease Control andPrevention; Parasites-African trypanosomiasis, available athttp://www.cdc.gov/parasites/sleeping.sickness/biology.html). Seventyanimal species, including humans, have been found as natural reservoirhosts of Leishmania parasites (World Health Organization, Leishmaniasis,available at http://www.who.int/mediacentre/factsheets/fs375/en/).

At present, few drugs are available to treat or prevent human and animaldiseases caused by trypanosomatids. The available treatments often havesignificant toxicity issues, are often ineffective due to growingresistance, and are difficult to administer in the rural settings thatcharacterize many of the endemic areas. For these reasons, the WorldHealth Organization and the U. S. Centers for Disease Control andPrevention have targeted these diseases as priorities for new drugdevelopment.

Some flavonoids are known to exhibit a wide range of biologicalactivities, but the aurones, a small subset of the flavonoid family,have only more recently begun to attract much attention (Haudecoeur etal., Curr. Med. Chem. (2012) 19:2861-2875; Zwergel et al., Nat. Prod.Comm. (2012) 7:389-394). Aurones have been found in plants which produceyellow flowers and they play an important part in this yellowcoloration; however, aurones are found only in limited quantities, whichhas hindered exploration of their biological potential. Aurones displaya range of biological activities, including anti-cancer, anti-bacterial,anti-fungal, and anti-malarial activities (Carrasco et al., 2014, Eur.J. Med. Chem. 80:523-534; Demirayak et al., 2015, J. Enzyme Inhib. Med.Chem., p. 1-10; Song et al., 2015, Zhongguo Zhong Yao Za Zhi, v. 40, p.1097-101; Tiwari et al., 2012, Dalton Trans, 41:6451-7).

With respect to trypanosomal and related diseases, comparatively littlehas been reported. Ameta and co-workers reported the synthesis andevaluation of some highly functionalized aurones via an unusualcopper-mediated cyclization of a chalcone to an aurone (Ameta et al.,Int. J. Org. Chem. (2012) 2:295-301). The growth inhibition ofTrypanosoma cruzi at 10 μg/mL was modest, and cytotoxicity wassignificant.

More work has been reported in the area of Leishmaniasis. Early work byKayser and co-workers focused largely on compounds isolated from naturalsources (Kayser et al., Zeit. Naturforsch. C, 2002, 57, 717-720; Kayseret al., Tokai Journal of Experimental and Clinical Medicine. 1998, 23,423-426; Kayser et al., Planta Medica (1999) 65:316-319). More recently,Detsi and co-workers have prepared a number of aurone derivatives via aclosure of chalcones employing highly toxic mercuric acetate in pyridineat high temperatures (Roussaki et al., Int. J. Med. Chem. (2012) ArticleID 196921). Of the 12 compounds prepared, only half showed IC₅₀ valuesbelow 20 μM, with highly variable selectivity indices. Much like theKayser studies, these synthetic compounds were typically confined tooxygenated derivatives and two chlorinated derivatives, neither of whichwas particularly active.

Finally, the bioactivity of aurones against malaria has been explored insome of the Kayser papers cited herein, as well as two more recentpublications. Boumendjel and co-workers explored a series of aurones andaza-aurones, in which the oxygen of the coumaranone portion is replacedby a nitrogen (Haudecoeur et al., Curr. Med. Chem. (2012) 19:2861-2875;Souard et al., Bioorg. & Med. Chem. (2010) 5724-5731). The aza-auronesgenerally displayed better levels of activity, although still less thanthat displayed by chloroquine (a known anti-malarial). Moreira andco-workers studied a much broader range of aurone derivatives (Marta etal.; Eur. J. Med. Chem. 80 (2014) 523-534). The most effective compoundexhibited a low μM IC₅₀ value and high selectivity (SI>85), but was noteasy to synthesize. Interestingly, it appears to inhibit ABCtransporters, a family of proteins involved in multi-drug resistance.Synergy of this aurone and chloroquine was demonstrated.

Summary

Members of the library of compounds synthesized in Example I wereevaluated for anti-trypanosomal activity and mammalian cell toxicity.When compared with the basic aurone scaffold 53% of derivatives (44/83)showed improved activity against Trypanosoma brucei, the causative agentof African Sleeping Sickness. In addition, 27.7% of the compounds hadselectivity multiples (parasite inhibition vs. mammalian cell toxicity)greater than 10. These data demonstrate that aurone-based compounds havestrong potential for development of anti-trypanosomal therapies. See,e.g., Stubblefield et al., Planta Med 2016; 82—PC77, DOI:10.1055/s-0036-1578779.

Results

Activity, Toxicity, and Selectivity.

The anti-trypanosomal activity and mammalian cell toxicity of the basicaurone scaffold and the synthesized derivatives were assayed usingassays validated by Bowling as described in detail below (Bowling etal., Int J Parasit Drugs Drug Resist, 2012, 2, 262-270). Structureactivity relationship (SAR) data including mean percent inhibition at 50μM, IC₅₀ values and selectivity multiples for T. brucei, T. cruzi and L.amazonensis, and the mammalian cell toxicity model (L6) for all samplesare shown in Table 1 (ND=not determined).

TABLE 1 Structure Activity Relationships of Aurone-based Compounds. IC50(uM)^(a) Toxicity Selectivity ID Structure T. brucei T. cruzi L. amaz.(L6) L6/Tb L6/Tc L6/La Scaffold A.

Scaffold A- Unsubstituted 6615

40.09 15.78 4.13 329.43 8.22 20.88 79.77 Scaffold A- R1: MeO & OHSubstitutions 6601

40.28 10.78 3.30 >100 >2.48 >9.28 >30.30 6620

<1 35.09 <1^(c) >100 >100 >2.85 >100 2011

22.87 21.94 1.01 51.94 2.27 2.37 51.43 2001

22.35 >50 13.33 >100 >4.47 ND >7.50 2912

>50 >50 25.72 >100 ND ND >3.89 4004

19.69 >50^(b) 4.04 43.86 2.23 ND 10.86 2002

>50 >50 12.55 >100 ND ND >7.97 4003

27.68 >50 13.43 >100 >3.61 ND >7.45 9088

>50 18.41 9.54 >100 ND >5.43 >10.48 9252

34.94 16.53 1.52 >100 >2.86 >6.05 >65.79 9068

>50 >50 1.57 >200 ND ND >127.39 4005

>50 >50^(b) 3.58 >100 ND ND >27.93 9055

>50 >50 18.48^(c) >100 ND ND >5.41 9078

41.18 ND 1.45 70.71 1.72 ND 48.77 2911

>50 >50 6.51 52.64 ND ND 8.09 1009

18.83 >50 9.63 44.97 2.39 ND 4.67 9053

>50 >50 2.15 >200 ND ND >93.02 Scaffold A- R1: F Substitutions 9004

>50 22.00 10.46^(c) 175.99 ND 8.00 16.83 9024

>50 6.67 20.10 >100 ND <14.99 >4.98 9002

>50 15.79 39.34^(c) >100 ND >6.33 >2.54 Scaffold A- R1: Cl Substitutions9007

11.30 12.69 17.53^(c) 237.57 21.02 18.72 13.55 9026

27.42 10.96 12.61 77.49 2.83 7.07 6.15 9019

34.72 5.53 41.54 >100 >2.88 >18.08 >2.41 Scaffold A- R1: I Substitutions9003

>50 15.39 ND >100 ND >6.50 ND 9028

>50 7.54 2.61 >100 ND >13.26 >38.31 9029

>50 11.06 3.11 >100 ND >9.04 >32.15 Scaffold A- R1: Br Substitutions9006

28.32 21.31 <1^(c) 226.08 7.98 10.61 >226.08 9030

>50 7.35 <1^(c) >100 ND >13.61 >100 2009

41.71 11.41 10.29 >100 >2.40 >8.76 >9.72 1005

47.96 >50 31.07 >100 >2.09 ND >3.22 5005

47.20 >50 14.28 180.83 3.83 ND 12.66 6002

42.02 >50^(b) 11.03 >100 >2.38 ND >9.07 4002

42.82 >50^(b) 19.74 >100 >2.34 ND >5.07 2905

>50 >50 38.97 >100 ND ND >2.57 9056

21.92 35.97 1.83 >100 >4.56 >2.78 >54.64 3003

>50 >50 16.02 >100 ND ND >6.24 3012

>50 40.11^(b) 1.21 >100 ND >2.49 >82.64 Scaffold A- R1: CF3Substitutions 9086

37.59 18.53 1.90 130.30 3.47 7.03 68.58 9085

>50 11.10 4.43 73.42 ND 6.61 16.57 9084

20.37 9.38 2.94 >400 >19.64 >42.64 >136.05 4006

30.21 15.80^(b) 9.70 72.94 2.41 4.62 7.52 2909

>50 >50 3.97 >100 ND ND >25.19 6001

>50 >50^(b) 7.29 >100 ND ND >13.72 Scaffold A- R1: Methyl Substitutions9057

24.36 26.34 <1 99.56 4.09 3.78 >99.56 9064

32.57 18.40^(b) 4.40 79.89 2.45 4.34 18.16 9065

>50 8.63 1.37 >100 ND >11.59 >72.99 5002

40.66 >50 10.53 183.07 4.50 ND 17.39 2904

>50 >50 5.63 70.27 ND ND >12.48 7000

23.18 7.92 ND 142.23 6.14 17.96 ND 6000

>50 >50 8.43 >100 ND ND >11.86 Scaffold A- R1: NO2 Substitutions 2015

29.23 22.57 1.71 226.06 7.73 10.02 132.20 2010

40.73 19.96 14.51 248.98 6.11 12.47 17.16 7001

>50 >50 22.01^(c) >100 ND ND >4.54 Scaffold A- R1: CN Substitutions 3007

>50 46.63 17.56 >100 ND >2.14 >5.69 9070

34.68 16.74 <1 >200 >5.77 >11.95 >200 2014

8.12 23.52 10.73 239.18 29.46 10.17 22.29 6003

17.10 >50^(b) 1.22 >100 >5.85 ND >81.97 9076

>50 26.29 2.44 >100 ND >3.80 >40.98 5006

30.29 >50 4.79 213.19 7.04 ND 44.51 4001

2.66 31.71^(b) 38.43 76.24 28.66 2.40 1.98 7002

>50 >50^(b) 2.08^(c) 82.54 ND ND 39.68 Scaffold A- R1: Misc.Substitutions 8001

46.53 11.39 6.42^(c) >100 >2.15 >8.78 >15.58 8002

43.40 16.65 2.88^(c) 89.04 2.05 5.35 30.92 3008

>50 >50 3.57 99.28 ND ND 27.81 3009

>50 >50 2.36 35.03 ND ND 14.84 9087

39.03 5.38 1.65 144.34 3.70 26.83 87.48 2018

>50 34.42 7.22 298.28 ND 8.67 41.31 9047

>50 >50 24.20^(c) >100 ND ND >4.13 3001

>50 >50 <1 >100 ND ND >100 3006

>50 >50 12.52 >100 ND ND >7.99 9063

>50 18.90 1.13 94.04 ND 4.98 83.22 Scaffold A- R1: C SubstitutionsScaffold A- R1: Pyridine Substitution 9051

23.15 19.37 6.26 79.58 3.44 4.11 12.71 2008

>50 16.44 26.29^(c) 81.06 ND 4.93 3.08 3011

>50 32.94 2.61 >100 ND >3.04 >38.31 9260

43.61 14.56 ND >100 >2.29 >6.87 ND 9253

>50 17.81 7.38 <100 ND >5.61 >13.55 9312

>50 >50^(b) <1 >200 ND ND >200 Scaffold B- R1: C substitutions with O

2023

>50 6.97 <1 >100 ND >14.35 >100 3005

41.89 7.47 42.74 >100 >2.39 >13.39 >2.34 9067

34.94 12.49 <1 31.05 0.89 2.49 >31.05 9251

35.33 3.57 2.36 85.46 2.42 23.94 36.21 3002

20.18 6.44 6.48 >100 >4.96 >15.53 >15.43 TA2

11.43 >50^(b) <1 >200 >17.50 ND >200 Scaffold B- R1: C substitutionswith NH

2906

34.63 >50 19.87^(c) >100 >2.89 ND >5.03 2021

38.86 14.63 <1 82.71 2.13 5.65 >82.71 9062

24.84 8.53 1.66 64.71 2.61 7.59 38.98 2026

16.07 20.91 <1 >100 >6.22 >4.78 >100 6621

1.56 18.34 >50 >100 >64.10 >5.45 ND 9059

>50 5.25 3.96 320.73 ND 61.09 80.99 Scaffold B- R1: C substitutions withS

1001

25.28 >50 22.93 50.56 2.00 ND 2.20 2901

>50 >50 19.37 >100 ND ND >5.16 5001

>50 >50 21.12 >100 ND ND >4.73 3004

38.71 9.02 24.22 >100 >2.58 >11.09 >4.13 2004

41.78 13.65 <1 >100 >2.39 >7.33 >100 9050

>50 >50 9.24 >100 ND ND >10.82 9058

>50 34.82 2.73 79.31 ND 2.28 29.05 9060

>50 34.62 1.36 >100 ND >2.89 >73.53 9061

>50 >50 1.70 >100 ND ND >58.82 Scaffold B- R1: Misc. 6617

35.36 39.15 3.62 >400 >11.31 >10.22 >110.50 2013

13.19 14.06 3.03^(c) 57.97 4.39 4.12 19.13 Scaffold C- Aza-aurones AA3A

>50 >50 <1 >200 ND ND >200 AA4A

34.69 >50^(b) <1^(c) 86.95 2.51 ND >86.95 AA5

49.33 27.80 ND 39.74 0.81 1.43 ND AA5A

26.31 33.51 <1 39.76 1.51 1.19 >39.76 AA8

11.72 16.78 <1 >100 >8.53 >5.96 >100 AA9

>50 >50 <1 >200 ND ND >200 AA11

>50 >50^(c) 1.86 >200 ND ND >107.53 ^(a)Mean of 2(+) independent trailsexcept as noted. T. cruzi values from intracellular assay except asnoted; ^(b)Extracellular T. cruzi assay; ^(c)Value obtained from onetrial.

The derivatives produced a broad range of activity in both T. brucei andL6. Dose response assays were used to determine the minimum dose thatproduced 50% inhibition (IC₅₀). These data demonstrate that a variety ofsubstitutions on the base aurone scaffold (compound 6615) are effectivein increasing anti-trypanosomal activity.

The most promising compounds are those that demonstrate high parasiteinhibition (lowest IC₅₀ values) and low toxicity (highest IC₅₀ values inthe L6 assay). Compounds with the strongest antitrypanosomal activity(IC₅₀<10 μM) against T. brucei, T. cruzi, and L. amazonensis are shownin Table 2 (ND=not determined). These compounds include compounds 6620,6621, 4001, 2014, 9251, 9059, 9087, 9019, 3002, 9024, 2023, 9030, 3005,9028, 7000, 9062, 9065, 3004, 9084, 9067, 2004, 2021, 9070, AA8, 2026,9006, 9057, AA5A, TA2, AA4A, 3001, 9312, AA3A, AA9, 2011, 9063, 3012,6003, 9060, 9078, 9252, 9068, 9061, 2015, 9056, AA11, 9086, 7002, 9053,3009, 9076, 3011, 9058, 8002, 2013, 9029, 6601, 3008, 4005, 6617, 2909,4004, 9064, 9085, 5006, 2904, 9051, 8001, 2911, 2018, 6001, 9253, 6000,9050, 9088, 1009, and 4006. Some substituted aurones, such as compounds2023, 3002, 6620, 9028, 9030, 9059, 9062, 9065, 9084, 9087, and 9251,are particularly preferred because they are useful to treat two or moretrypanosomid infections. Other useful substituted aurones includecompounds 2001, 9007, 2008, 2906, and 1001.

TABLE 2 Compounds with Strongest Antitrypanosomal Activity andSelectivity IC₅₀ (uM) ^(a) Toxicity Selectivity ID T. brucei T. cruzi L.amaz. (L6) L6/Tb L6/Tc L6/La 6615 40.09 15.78 4.13 329.43 8.22 20.887977 Compounds with strongest effect vs. T. brucei 6620 <1 35.09 <1^(c) >100 >100 >2.85 >100 6621 1.56 18.34 >50 >100 >64.10 >5.45 ND 40012.66 31.71 ^(b) 38.43 76.24 28.66 2.40 1.98 2014 8.12 23.52 10.73 239.1829.46 10.17 22.29 Compounds with strongest effect vs. T. cruzi 925135.33 3.57 2.36 85.46 2.42 23.94 36.21 9059 >50 5.25 3.96 320.73 ND61.09 80.99 9087 39.03 5.38 1.65 144.34 3.70 26.83 87.48 9019 34.72 5.5341.54 >100 >2.88 >18.08 >2.41 3002 20.18 6.446.48 >100 >4.96 >15.53 >15.43 9024 >50 6.67 20.10 >100 ND <14.99 >4.982023 >50 6.97 <1 >100 ND >14.35 >100 9030 >50 7.35 <1 ^(c) >100ND >13.61 >100 3005 41.89 7.47 42.74 >100 >2.39 >13.39 >2.34 9028 >507.54 2.61 >100 ND >13.26 >38.31 7000 23.18 7.92 ND 142.23 6.14 17.96 ND9062 24.84 8.53 1.66 64.71 2.61 7.59 38.98 9065 >50 8.63 1.37 >100ND >11.59 >72.99 3004 38.71 9.02 24.22 >100 >2.58 >11.09 >4.13 908420.37 9.38 2.94 >400 >19.64 >42.64 >136.05 Compounds with strongesteffect vs. L. amazonensis 2023 >50 6.97 <1 >100 ND >14.35 >100 9030 >507.35 <1 ^(c) >100 ND >13.61 >100 9067 34.94 12.49 <1 31.05 0.892.49 >31.05 2004 41.78 13.65 <1 >100 >2.39 >7.33 >100 2021 38.86 14.63<1 82.71 2.13 5.65 >82.71 9070 34.68 16.74 <1 >200 >5.77 >11.95 >200 AA811.72 16.78 <1 >100 >8.53 >5.96 >100 2026 16.07 20.91<1 >100 >6.22 >4.78 >100 9006 28.32 21.31 <1 ^(c) 226.08 7.9810.61 >226.08 9057 24.36 26.34 <1 99.56 4.09 3.78 >99.56 AA5A 26.3133.51 <1 39.76 1.51 1.19 >39.76 6620 <1 35.09 <1^(c) >100 >100 >2.85 >100 TA2 11.43 >50 ^(b) <1 >200 >17.50 ND >200 AA4A34.69 >50 ^(b) <1 ^(c) 86.95 2.51 ND >86.95 3001 >50 >50 <1 >100 NDND >100 9312 >50 >50 ^(b) <1 >200 ND ND >200 AA3A >50 >50 <1 >200 NDND >200 AA9 >50 >50 <1 >200 ND ND >200 2011 22.87 21.94 1.01 51.94 2.272.37 51.43 9063 >50 18.90 1.13 94.04 ND 4.98 83.22 3012 >50 40.11 ^(b)1.21 >100 ND >2.49 >82.64 6003 17.10 >50 ^(b) 1.22 >100 >5.85 ND >81.979060 >50 34.62 1.36 >100 ND >2.89 >73.53 9065 >50 8.63 1.37 >100ND >11.59 >72.99 9078 41.18 ND 1.45 70.71 1.72 ND 48.77 9252 34.94 16.531.52 >100 >2.86 >6.05 >65.79 9068 >50 >50 1.57 >200 ND ND >127.39 908739.03 5.38 1.65 144.34 3.70 26.83 87.48 9062 24.84 8.53 1.66 64.71 2.617.59 38.98 9061 >50 >50 1.70 >100 ND ND >58.82 2015 29.23 22.57 1.71226.06 7.73 10.02 132.20 9056 21.92 35.97 1.83 >100 >4.56 >2.78 >54.64AA11 >50 >50 ^(c) 1.86 >200 ND ND >107.53 9086 37.59 18.53 1.90 130.303.47 7.03 68.58 7002 >50 >50 ^(b) 2.08 ^(c) 82.54 ND ND 39.689053 >50 >50 2.15 >200 ND ND >93.02 9251 35.33 1.57 2.36 85.46 2.4223.94 36.21 3009 >50 >50 2.36 35.03 ND ND 14.84 9076 >50 26.29 2.44 >100ND >3.80 >40.98 9028 >50 7.54 2.61 >100 ND >13.26 >38.31 3011 >50 32.942.61 >100 ND >3.04 >38.31 9058 >50 34.82 2.73 79.31 ND 2.28 29.05 800243.40 16.65 2.88 ^(c) 89.04 2.05 5.35 30.92 9084 20.37 9.382.94 >400 >19.64 >42.64 >136.05 2013 13.19 14.06 3.03 ^(c) 57.97 4.394.12 19.13 9029 >50 11.06 3.11 >100 ND >9.04 >32.15 6601 40.28 10.783.30 >100 >2.48 >9.28 >30.30 3008 >50 >50 3.57 99.28 ND ND 27.814005 >50 >50 ^(b) 3.58 >100 ND ND >27.93 6617 35.36 39.153.62 >400 >11.31 >10.22 >110.50 9059 >50 5.25 3.96 320.73 ND 61.09 80.992909 >50 >50 3.97 >100 ND ND >25.19 4004 19.69 >50 ^(b) 4.04 43.86 2.23ND 10.86 9064 32.57 18.40 ^(b) 4.40 79.89 2.45 4.34 18.16 9085 >50 11.104.43 73.42 ND 6.61 16.57 5006 30.29 >50 4.79 213.19 7.04 ND 44.512904 >50 >50 5.63 70.27 ND ND >12.48 9051 23.15 19.37 6.26 79.58 3.444.11 12.71 8001 46.53 11.39 6.42 ^(c) >100 >2.15 >8.78 >15.58 3002 20.186.44 6.48 >100 >4.96 >15.53 >15.43 2911 >50 >50 6.51 52.64 ND ND 8.092018 >50 34.42 7.22 298.28 ND 8.67 41.31 6001 >50 >50 ^(b) 7.29 >100 NDND >13.72 9253 >50 17.81 7.38 <100 ND >5.61 >13.55 6000 >50 >508.43 >100 ND ND >11.86 9050 >50 >50 9.24 >100 ND ND >10.82 9088 >5018.41 9.54 >100 ND >5.43 >10.48 1009 18.83 >50 9.63 44.97 2.39 ND 4.674006 30.21 15.80 ^(b) 9.70 72.94 2.41 4.62 7.52 Compounds with IC₅₀ <10uM on 2(+) parasites 2023 >50 6.97 <1 >100 ND >14.35 >100 3002 20.186.44 6.48 >100 >4.96 >15.53 >15.43 6620 <1 35.09 <1^(c) >100 >100 >2.85 >100 9028 >50 7.54 2.61 >100 ND >13.26 >38.319030 >50 7.35 <1 ^(c) >100 ND >13.61 >100 9059 >50 5.25 3.96 320.73 ND61.09 80.99 9062 24.84 8.53 1.66 64.71 2.61 7.59 38.98 9065 >50 8.631.37 >100 ND >11.59 >72.99 9084 20.37 9.382.94 >400 >19.64 >42.64 >136.05 9087 39.03 5.38 1.65 144.34 3.70 26.8387.48 9251 35.33 3.57 2.36 85.46 2.41 23.94 36.21 Compounds withselectivity >10X 2004 41.78 13.65 <1 >100 >2.39 <7.33 >100 2010 40.7319.96 14.51 248.98 6.11 12.47 17.16 2011 22.87 21.94 1.01 51.94 1.172.37 51.43 2013 13.19 14.06 3.03 ^(c) 57.97 4.39 4.12 19.13 2014 8.1223.52 10.73 239.18 29.46 10.17 22.29 2015 29.23 22.57 1.71 226.06 7.7310.02 132.20 2018 >50 34.42 7.22 298.28 ND 8.67 41.31 2021 38.86 14.63<1 82.71 2.13 5.65 >82.71 2023 >50 6.97 <1 >100 ND >14.35 >100 202616.07 20.91 <1 >100 >6.22 >4.78 >100 2904 >50 >50 5.63 70.27 NDND >12.48 2909 >50 >50 3.97 >100 ND ND >25.19 3001 >50 >50 <1 >100 NDND >100 3002 20.18 6.44 6.48 >100 >4.96 >15.53 >15.43 3004 38.71 9.0224.22 >100 >2.58 >11.09 >4.13 3005 41.89 7.4742.74 >100 >2.39 >13.39 >2.34 3008 >50 >50 3.57 99.28 ND ND 27.813009 >50 >50 2.36 35.03 ND ND 14.84 3011 >50 32.94 2.61 >100ND >3.04 >38.31 3012 >50 40.11 ^(b) 1.21 >100 ND >2.49 >82.64 400419.69 >50 ^(b) 4.04 43.86 2.23 ND 10.86 4005 >50 >50 ^(b) 3.58 >100 NDND >27.93 5002 40.66 >50 10.53 183.07 4.50 ND 17.39 5005 47.20 >50 14.28180.83 3.83 ND 12.66 5006 30.29 >50 4.79 213.19 7.04 ND 44.516000 >50 >50 8.43 >100 ND ND >11.86 6001 >50 >50 ^(b) 7.29 >100 NDND >13.72 6003 17.10 >50 ^(b) 1.22 >100 >5.85 ND >81.97 6601 40.28 10.783.30 >100 >2.48 >9.28 >30.30 6617 35.36 39.153.62 >400 >11.31 >10.22 >110.50 6620 <1 35.09 <1^(c) >100 >100 >2.85 >100 6620 <1 35.09 <1 ^(c) >100 >100 >2.85 >1006621 1.56 18.34 >50 >100 >64.10 >5.45 ND 7000 23.18 7.92 ND 142.23 6.1417.96 ND 7002 >50 >50 ^(b) 2.08 ^(c) 82.54 ND ND 39.68 8001 46.53 11.396.42 ^(c) >100 >2.15 >8.78 >15.58 8002 43.40 16.65 2.88 ^(c) 89.04 2.055.35 30.92 9004 >50 22.00 10.46 ^(c) 175.99 ND 8.00 16.83 9006 28.3221.31 <1 ^(c) 226.08 7.98 10.61 >226.08 9007 11.30 12.69 17.53 ^(c)237.57 21.02 18.72 13.55 9019 34.72 5.53 41.54 >100 >2.88 >18.08 >2.419024 >50 6.67 20.10 >100 ND <14.99 >4.98 9028 >50 7.54 2.61 >100ND >13.26 >38.31 9029 >50 11.06 3.11 >100 ND >9.04 >32.15 9030 >50 7.35<1 ^(c) >100 ND >13.61 >100 9050 >50 >50 9.24 >100 ND ND >10.82 905123.15 19.37 6.26 79.58 3.44 4.11 12.71 9053 >50 >50 2.15 >200 NDND >93.02 9056 21.92 35.97 1.83 >100 >4.56 >2.78 >54.64 9057 24.36 26.34<1 99.56 4.09 3.78 >99.56 9058 >50 34.82 2.73 79.31 ND 2.28 29.059059 >50 5.25 3.96 320.73 ND 61.09 80.99 9060 >50 34.62 1.36 >100ND >2.89 >73.53 9061 >50 >50 1.70 >100 ND ND >58.82 9062 24.84 8.53 1.6664.71 2.61 7.59 38.98 9063 >50 18.90 1.13 94.04 ND 4.98 83.22 9064 32.5718.40 ^(b) 4.40 79.89 2.45 4.34 18.16 9065 >50 8.63 1.37 >100ND >11.59 >72.99 9067 34.94 12.49 <1 31.05 0.89 2.49 >31.05 9068 >50 >501.57 >200 ND ND >127.39 9070 34.68 16.74 <1 >200 >5.77 >11.95 >2009076 >50 26.29 2.44 >100 ND >3.80 >40.98 9078 41.18 ND 1.45 70.71 1.72ND 48.77 9084 20.37 9.38 2.94 >400 >19.64 >42.64 >136.05 9085 >50 11.104.43 73.42 ND 6.61 16.57 9086 37.59 18.53 1.90 130.30 3.47 7.03 68.589087 39.03 5.38 1.65 144.34 3.70 26.83 87.48 9088 >50 18.41 9.54 >100ND >5.43 >10.48 9251 35.33 3.57 2.36 85.46 2.42 23.94 36.21 9252 34.9416.53 1.52 >100 >2.86 >6.05 >65.79 9253 >50 17.81 7.38 <100ND >5.61 >13.55 9312 >50 >50 ^(b) <1 >200 ND ND >200 AA11 >50 >50 ^(c)1.86 >200 ND ND >107.53 AA3A >50 >50 <1 >200 ND ND >200 AA4A 34.69 >50^(b) <1 ^(c) 86.95 2.51 ND >86.95 AA5A 26.31 33.51 <1 39.76 1.511.19 >39.76 AA8 11.72 16.78 <1 >100 >8.53 >5.96 >100 AA9 >50 >50 <1 >200ND ND >200 TA2 11.43 >50 ^(b) <1 >200 >17.50 ND >200 ^(a) mean of 2(+)independent trails except as noted. T cruzi values from intracellularassay except as noted; ^(b) Extracellular T. cruzi assay; ^(c) Valueobtained from one trial.

Structure/Activity Analysis of Selected Aldehyde-Based Substitutions.

The aldehyde derived fragment of the aurone scaffold was explored, asdescribed in Example III, to determine modifications which mightincrease antitrypanosomal activity. Derivatives were synthesized to addvarious substituents to the aldehyde-derived fragment as shown in Table3A: halogens (2a-6c), cyano groups, which are bioisosteres of the CF3s(7a-c), groups that were likely to affect lipophilicity (8a-9b), strongelectron withdrawing groups (10a-11), and combinations of methoxy andhydroxyls mimicking those that might be found in nature (14a-16c).Replacement of the aldehyde-derived ring with 5- and 6-memberedheteroaromatic rings containing O, NH, and S are shown in Table 3B.Additional miscellaneous substitutions are shown in Table 3C.

TABLE 3A Antitrypanosomal activity of aldehyde-based substitutions.

Selectivity^(b) Series Sample IC50 (uM)^(a) L6/ L6/ ID ID SubstitutionTB SEM TC-I SEM L6 SEM Tb TC-I 1 6615 — 40.09 1.12 15.78 3.30 329.434.99 8.22 20.88 2a 9004 2 F >50 22.00 8.45 175.99 5.19 * 8.00 2b 9024 3F >50 6.67 3.76 >100 * 14.99 2c 9002 4 F >50 15.79 3.29 >100 * >6.33 3a9007 2 Cl 11.30 0.16 12.69 2.33 237.57 10.72 21.02 18.72 3b 9026 3 Cl27.42 1.00 10.96 2.15 77.49 4.01 2.83 7.07 3c 9019 4 Cl 34.72 0.35 5.530.74 >100 >2.88 >18.08 4a 9003 2 I >50 15.39 4.57 >100 * >6.50 4b 9028 3I >50 7.54 1.54 >100 * >13.26 4c 9029 4 I >50 11.06 1.88 >100 * >9.04 5a9006 2 Br 28.32 0.87 21.31 2.97 226.08 3.95 7.98 10.61 5b 9030 3 Br >507.35 1.51 >100 * >13.61 5c 2009 4 Br 41.71 1.93 11.411.16 >100 >2.40 >8.76 5d 3003 2, 5 Br >50 >50 >100 * * 5e 3012 2 OH, 3,5Br >50 >50 >100 * * 5f 9056 2 Br, 4,5 MeO 21.92 9.23 35.976.37 >100 >4.56 >2.78 6a 9086 2 CF3 37.59 1.60 18.53 2.61 130.30 4.253.47 7.03 6b 9085 3 CF3 >50 11.10 1.88 73.42 * 6.61 6c 9084 4 CF3 20.370.18 9.38 2.08 >400 >19.64 >42.64 7a 3007 2 CN >50 46.633.73 >100 * >2.14 7b 9070 3 CN 34.68 1.89 16.74 2.40 >200 >5.77 >11.957c 2014 4 CN 8.12 0.10 23.52 3.10 239.18 12.20 29.46 10.17 8a 9057 2 CH324.36 0.76 26.34 2.59 99.56 0.86 4.09 3.78 8b 9064 3 CH3 32.57 0.7037.90 10.29 79.89 0.62 2.45 2.11 8c 9065 4 CH3 >50 8.630.73 >100 * >11.59 9a 8001 4 Et 46.53 0.31 11.39 3.00 >100 >2.15 >8.789b 8002 4 iPr 43.40 0.27 16.65 0.51 89.04 1.09 2.05 5.35 10 3008 4tBu >50 12.51 2.35 99.28 0.80 * 7.94 11 3009 4 nBu >50 7.99 2.01 35.031.37 * 4.38 12a 2015 3 NO2 29.23 0.70 22.57 2.48 226.06 3.54 7.73 10.0212b 2010 4 NO2 40.73 0.69 19.96 1.28 248.98 2.96 6.11 12.47 13 9047 4CO2Me >50 >50 >100 * * 14a 6601 4 MeO 40.28 1.01 10.78 2.16 >1001.11 >2.48 >9.28 14b 2011 3,4 MeO 22.87 0.64 21.94 0.89 51.94 2.27 2.3714c 2001 2,3,4 MeO 22.35 2.31 >50 >100 >4.47 * 14d 2002 3,4,5MeO >50 >50 >100 * * 15a 9088 2 OH >50 18.41 2.46 >100 * >5.43 15b 92523 OH 34.94 0.17 16.53 2.54 >100 >2.86 >6.05 15c 9068 4OH >50 >50 >100 * * 16a 9055 2 OH, 3 MeO >50 >50 >100 * * 16b 9078 3 OH,4 MeO 41.18 0.11 ND^(c) 70.71 0.48 1.72 * 16c 9053 3 MeO, 4OH >50 >50 >200 * * ^(a)Weighted means of two or mote independenttrials. ^(b)Where the highest dose tested for L6 did not produceinhibition sufficient to calculate IC50, selectivity was estimated basedon the highest dose tested. ^(c)IC₅₀ could not be detetmined due to hostcell toxicity.

TABLE 3B Antitrypanosomal activity of five-membered heteroaromatic ringsubstitutions. Selectivityb Series Samplc IC50 (μM)a L6/ L6/ ID ID TBSEM TC-I SEM L6 SEM Tb Tc-I 17a 9051

23.15 0.63 19.37 3.00 79.58 0.83 3.44 4.11 17b 3011

>50 32.94 3.52 >100 * >3.04 17c 9260

43.61 0.00 14.56 1.49 >100 >4.96 >6.87 17d 9253

>50 17.81 5.26 >100 * >5.61 17e 2008

>50 16.44 0.93 81.06 1.07 * 4.93 18a 2023

>50 6.97 1.52 >100 * >14.35 18b 9067

34.94 0.72 12.49 1.91 31.05 0.26 −0.89 2.49 18c 9251

35.33 0.77 3.57 0.70 85.46 0.67 2.42 3.94 18d 3005

41.89 0.29 7.47 1.92 >100 >2.39 >13.39 18e 3002

20.18 0.17 6.44 1.59 >100 >4.96 >15.53 19a 2021

38.86 0.34 14.63 1.12 82.71 0.72 2.13 5.65 19b 9062

24.84 0.66 8.53 2.12 64.71 3.71 2.61 7.59 19c 9063

>50 18.90 2.67 94.04 0.56 * 4.98 20a 6621

>50 18.34 2.18 >100 * >5.45 20b 2026

16.07 0.22 20.91 6.06 >100 >6.22 >4.78 20c 9059

>50 5.25 2.02 320.73 5.22 * 61.09 21a 2004

41.78 0.31 13.65 1.50 >100 >2.39 >7.33 21b 9050

>50 >50 >100 * * 21c 9058

>50 34.82 9.59 79.31 0.20 * 2.28 21d 9060

>50 34.62 4.15 >100 * >2.89 21e 9061

>50 >50 >100 * * 21f 3004

38.71 0.45 9.02 1.48 >100 >2.58 >11.09 aWeighted means of two or moreindependent trials. bWhere the highest dose tested for L6 did notproduce inhibition sufficient to calculate IC50, selectivity wasestimated based on the highest dose tested.

TABLE 3C Antitrypanosomal activity of miscellaneous substitutions.Selectivityb Series Sample IC50 (μM)a L6/ L6/ ID ID Structure TB SEMTC-I SEM L6 SEM Tb TC-I 22 6617

35.36 1.13 39.15 6.09 >400 >11.31 >10.22 23 2013

13.19 0.12 14.06 2.49 57.97 0.58 4.39 4.12 24 9087

39.03 0.10 5.38 0.68 144.34 17.29 3.70 26.83 25 3001

>50 >50 >100 * * 26 3006

>50 >50 >100 * * 27 2018

>50 34.42 3.46 298.28 2.54 * 8.67 aWeighted means of two or moreindependent trials. bWhere the highest dose tested for L6 did notproduce inhibition sufficient to calculate IC50, selectivity wasestimated based on the highest dose tested.

These compounds exhibited a broad range of biological activity, with T.cruzi and T. brucei IC₅₀ doses as low as 3.57 μM (18c) and 8.12 μM (7c),respectively. Most of the aurone analogs demonstrate fairly modesttoxicity using the L6 model, thus leading to therapeutically usefullevels of selectivity with highest selectivity of 61.09 (20c) and 29.49(7c), for T. cruzi and T. brucei, respectively. The nature and positionof functional groups on the aldehyde derived portion of these auroneplays a role in determining their activity.

In general, halogens (2a-6c) were quite effective against T. brucei,with chlorine and bromine being the best and bromine being much moreposition sensitive. While F substitutions were ineffective at anyposition, CF3 substitutions were more successful and likewise positionsensitive. Interestingly, the addition of CN (7a-c), a bioisostere ofCF3, produced the strongest effect against T. brucei of any auronetested (7c IC50=8.12 μM). The halogens were also effective against T.cruzi, with Cl, I, Br, and CF3 substitutions producing four compoundswith IC50 doses <10 μM. While CN substitutions were active against T.cruzi they did not show increased effect over CF3s similar to theresults seen with T. brucei.

Rather surprisingly, alkyl groups (8a-11) were generally beneficial. ForT. brucei, Me additions were favorable while increasing chain length(Et, iPr, butyl (n or tert)) decreased activity. This group also showedstrong, position sensitive activity against T. cruzi with two compoundsproducing IC50 doses <10 μM. While 8a (2 CH3) and 8b (3 CH3) producedsimilar results for both T. brucei and T. cruzi, 8c (3 CH3) waseffective against T. cruzi only. In T. cruzi, the increased chain lengthand corresponding lipophilicity also showed strong effect.

In order to probe whether the influence of CN substitution (7a-c) waspurely electronic or perhaps also had a geometric component, nitro (12aand 12b) and ester (13) containing compound were also explored. Whilethe two nitro compounds did display modest activity against T. cruzi andT. brucei, the ester compound displayed no activity, thus indicatingthat geometric considerations are important.

Oxygenated systems (14a-16c) were not as effective, which is interestingas naturally occurring aurones are highly oxygenated. The T. cruzi IC50dose for 16b could not be determined due to the high level of host celltoxicity.

Heteroaromatic aldehydes (Table 3B) afforded aurones with modest levelsof activity for T. brucei in virtually all cases with the exception ofhalogenated thiophenes, but none were as good as the benzenoidcompounds.

However, the heteroaromatic substitutions produced seven compounds withT. cruzi IC50 doses <10 μM. Pyridine substitutions (17a-e) produced twocompounds, 17a and 17c, with moderate activity against T. brucei (IC50doses of 23.15 and 43.61 μM, respectively). All five compounds showedmoderate activity against T. cruzi with IC50 doses ranging from14.56-32.94 μM.

The replacement of the six-membered aldehyde-derived ring with afive-membered furan (18a-e) proved very effective against T. cruzi withall five compounds producing T. cruzi IC50 doses <12.5 μM. Compound 18cproduced the strongest effect against T. cruzi (IC50=3.57 μM) of anyaurone tested in this study. While not showing the strongest effect inthis group, it is interesting to note that 18b, which produced a T.cruzi IC50 of 12.49 μM, has also been shown to have stronganti-inflammatory activity. 20 However, the toxicity in L6 for 18b(31.05 μM) and 18c (85.46 μM) resulted in low selectivity. All but oneof the compounds in this series, 18a produced moderate activity in T.brucei as well with 1050 doses ranging from 20.18-35.53 μM.

The most successful heteroaromatic strategy for T. brucei was 20b(2026z), an imidazole substitution which produced an IC50 dose of 16.07μM and selectivity >6.22. Within the group of five-membered rindnitrogen-containing compounds (19a-20c), only the pyrrole and oneimidazole, 19a-b, had moderate activity for T. brucei. However, thisgroup was much more successful for T. cruzi, with all six compounds inthis group producing strong activity (IC50 doses <21 μM) and two of thesix having IC50 doses <10 μM. The most promising aurone in this studyfor T. cruzi in terms of both antitrypanosomal effect and selectivitywas the methylated imidazole, 20c, which produced an 1050 of 5.25 μM anda selectivity of 61.09.

Thiophene substitutions (21a-f) produced only two compounds withmoderate activity in T. brucei. These two compounds 21a and 21f,produced strong activity in T. cruzi, 13.65 and 9.02 μM, respectively.The brominated thiophenes (21b-21e) produced no activity in T. bruceiand moderate to no activity in T. cruzi.

Of the miscellaneous substitutions shown in Table 3C (22-27), the highlyelectron-rich compound 24 produced the strongest activity against T.cruzi with an IC50 dose of 5.38 μM and selectivity of 26.83. Cinnamatecompound 23 produced strong effects in both T. brucei and T. cruzi(13.19 and 14.06 μM, respectively), raising the broad family ofcinnamates as interesting candidates for future study, althoughsignificant toxicity is a concern.

Three different aldehyde-based substitution strategies produced thethree most effective compounds against T. brucei IC50<20 μM. The 4 CNsubstitution (7c) produced the strongest activity with an IC50 of 8.12μM and selectivity of 29.46. The 2 Cl substitution (3a) produced an IC50of 11.30 μM and selectivity of 21.02. The methylated imidazole (20b)produced an IC50 of 16.07 and selectivity of >6.22. All three of thesestrategies also produced activity in T. cruzi with IC50 doses rangingfrom 12.69-23.52 μM.

Multiple aldehyde-based substitution strategies were effective ingenerating the 14 compounds with T. cruzi IC50 doses <10 μM.Halogenation produced four compounds (3c, 4b. 5b, and 6c). Substitutionwith alkyl groups produced two compounds (8c and 11). Five-memberedheteroaromatic substitutions produced seven compounds (18a, 18c-e, 19b,20c, and 21f). Six of these compounds (4b, 5b, 8c, 11, 18a, and 20c)were selective for T. cruzi and produced insufficient activity at thehighest dose tested to determine an IC50 (IC50>50 μM). The remainingeight produced moderate activity produced activity that was more than 2×selective for T. cruzi (T. brucei IC50>20 μM).

These data demonstrate that aurone-based compounds have strong potentialfor development of anti-trypanosomal therapies.

Materials and Methods

Dried compounds were initially suspended in DMSO at concentrationsranging from 10-40 mM. Prior to assays compounds were further diluted infresh assay media to the desired concentrations.

T. brucei Culture and Assay

Trypanosoma brucei brucei 427 cells were maintained in HMI-9 mediumsupplemented with 10% heat inactivated fetal calf serum (AtlantaBiologicals, Atlanta, Ga.) and Penicillin-Streptomycin (P/S)(Penicillin, 5000 U/mL-Streptomycin, 5 mg/ml) purchased from Sigma (St.Louis, Mo.). The resazurin-based metabolic indicator, PrestoBlue, waspurchased from Invitrogen (Frederick, Md.)

Cells were passaged every 2-3 days and maintained in a 37° C. humidifiedincubator in an atmosphere of 5% CO₂. For assays, cells were countedusing a hemocytometer and adjusted using fresh media to deliver 5×10⁴cells per well (90 μL) in a translucent 96-well microtiter plate(Corning, Corning, N.Y.). Compound solutions diluted in culture mediumwere added (10 μL) to triplicate wells. Positive controls (cells treatedwith pentamidine (Sigma, St. Louis, Mo.)), negative controls (untreatedcells), solvent controls (DMSO), and media blanks were included on eachplate.

Following incubation for 48 h, 11 μL of PrestoBlue was added into eachwell. Relative fluorescence (RFU) readings were obtained afterincubation for an additional 24 h using excitation/emission setting of560/590 nm, using a SpectraMax M5 fluorescent plate reader (MolecularDevices, Sunnyvale, Calif.).

T. Cruzi Culture and Assay

Trypanosoma cruzi Tulahuen cells expressing beta-galactosidase (Buckneret al., Antimicrob. Agents Chemother. 40 (1996) 2592-2597) were culturedusing the L6 cell line as a host cell. Adherent L6 cultures wereinfected with freshly burst T. cruzi trypomastigotes. Approximately 4-6days later freshly burst trypomastigotes were collected and used tomaintain cultures For assays, trypomastigotes were centrifuged to aloose pellet and incubated for 3 hours to allow the trypomastigote formsto swim out of the pellet. Cells were adjusted for addition tointracellular or extracellular assays. For extracellular assays, cellswere adjusted to 45,000 cells per well and treated with compounds for 24hours at which time 100 uL of treated cultures were added 50 uL ofCellTiter Glo® (Promega) reagent. The resulting luminescence (or lackthereof) was quantified using a spectrophotometer and used to determineinhibition. For intracellular assays, cells were added to adherent L6cells using an infection ratio of 1:1 and immediately treated withcompounds. At 96 hours incubations, a solution containingchlorophenol-red-B-D-beta-galactopyranosidase (Sigma) and a lysis agentwere added. After an additional 2 hour incubation, a spectrophotometerwas used to quantify the resulting color change (or lack thereof) whichwas used to calculate inhibition.

L. amazonensis Culture and Assay

Leishmania amazonensis promastigotes expressing beta-lactamase (Buckneret al., Am. J. Trop. Med. Hyg. 72 (2005) 600-605. doi:72/5/600 [pii])were cultured in RPMI supplemented with 10% fetal calf serum and 1%pen-strep-glut solution (Sigma) at 27° C. Stationary phase promastigoteswere adjusted in fresh media, treated with compounds, and incubated at37° C. At 24 hours, 100 uL of cultures was added to 50 uL of theCellTiter Glo® solution. Luminescence readings were taken with aspectrophotometer and used to calculate inhibition.

L6 Culture and Assay

The rat skeletal muscle cell line, L6 (ATCC® CRL-1458) was obtained fromATCC as a model for mammalian toxicity. Cells were maintained in highglucose DMEM obtained from HyClone Laboratories (Logan, Utah) andsupplemented with FCS and P/S at the same concentrations used forparasite culture.

Cells were passaged every 3-4 days and maintained in a 37° C. humidifiedincubator in an atmosphere of 5% CO₂. For assays. cells were detached,counted using a hemocytometer, and adjusted using fresh media to deliver5×10³ cells per well (90 μL) in black walled, flat clear bottom 96-wellmicrotiter plates. After 3 h incubation for attachment, compoundsdiluted in culture medium were added (10 μL) to triplicate wells.Positive controls (cells treated with podophyllotoxin (Sigma, St. Louis,Mo.)), negative controls (untreated cells), solvent controls (DMSO) andmedia blanks were included on each plate.

Following incubation for 71.5 h, 11 μL of PrestoBlue was added into eachwell. RFU readings were obtained 30 min later using excitation/emissionsettings of 560/590 on the fluorescent plate reader.

Data Analysis

The percentage of cell inhibition was calculated using the formula:Percent Inhibition=1−((Treated Sample value−Medium onlyvalue)/(Untreated value−Medium only value))×100. Results are expressedas the mean of two or more independent trials. The minimum dose thatproduced 50% inhibition (IC₅₀) was calculated for each trial on GraphPadPrism software with a four parameter nonlinear regression.

Example III. Experimental Summary and Lead Drug Development

Most studies to date have targeted compounds closely related to thosefound in nature, which are highly oxygenated in both the benzofuranoneand aryl rings (Boumendjel et al., Chem. Pharm. Bull., 2002,50(6):854-6). The range of non-natural aurone derivatives that have beenprepared is fairly small. The recent development of a mild and efficientset of reaction conditions for the synthesis of aurones via thecondensation of a benzofuranone with an aldehyde by the Handy group hasenabled a more comprehensive study of aurone analogs, particularly thosewhich are less oxygenated (Hawkins et al., Tetrahedron 2013, 69 (44),9200-9204). As can be seen in the following sections, these lessoxygenated compounds display some very interesting levels of activityand represent a new and undeveloped area for novel anti-trypanosomaldrugs.

There are no known reports of the anti-trypanosomal properties ofaurone-based compounds. The aurone framework provides inherentlyinnovative features, including a simple skeleton devoid ofstereocenters, easy synthesis in 1-4 steps from commercially availablematerials, facile tuning of electronic and steric factors important forfuture SAR efforts, and an intrinsically drug-like character.

In our approach, the aurone can be viewed as coming from two halves thealdehyde derived fragment (ADF) and the benzofuranone derived fragment(BDF). (FIG. 2) Our first generation of derivatives (discussed in moredetail below) focused primarily on exploration of the ADF coupled withan unsubstituted BDF. A wide range of ADF, including ones that arehalogenated, alkyl substituted, oxygenated, and heteroaromatic, wereexamined.

These compounds demonstrated biological activity and chemical propertiesthat support the further exploration of this framework asanti-trypanosomal agents:

1. These compounds have anti-trypanosomal properties which can beoptimized with simple structural modifications. Compounds havingaldehyde-derived substitutions that showed the strongest parasiteinhibition for T. brucei T. cruzi, and L. amazonensis are among thoselisted in Table 2 (Example II).

2. The compounds have potential broad spectrum activity with a number ofcompounds showing activity against two or more parasites.

3. The compounds were generally non-toxic to the mammalian cell toxicitymodel (L6).

4. The compounds can be readily prepared in high purity and quantity.Our first generation of derivatives were synthesized in 1-4 steps andgenerated generally high yields (the majority were >50% underun-optimized conditions).

5. The compounds exhibit drug-like properties. The base aurone scaffoldobeys Lipinski's Rules of Five and other drug-like properties: MW is 222g/mol, octanol-water partition coefficient (clogP) is 3.20, and thetopological polar surface area (PSA) is 26.30 Å.

In the first generation of derivatives, the initial focus on the ADF wasbased upon the easy and inexpensive commercial availability of a widerange of aldehydes. While countless more aldehydes could be explored, anexamination of the data collected in terms of activity and selectivityso far shows that the most promising compounds are ones with the arylgroups shown in Table 2.

At the same time, comparatively little has been explored on the BDF. Inpart this is due to the more limited range of benzofuranones that arecommercially available at modest expense. Still, in the examples thathave been studied, we have noted that oxygenation (particularly hydroxylgroups) leads to decreased activity. This observation stands in contrastto most other aurone studies, which have focused on oxygenated compoundsmore closely related to naturally occurring aurones and have typicallyobserved improved activity, as in the anti-cancer studies of Boumenjdelet al. (Chem. Pharm. Bull., 2002, 50(6):854-6). In contrast, we haveobserved two substituted but not oxygenated benzofuranones (bromo andmethyl substituted) which have displayed either improved or similaractivity. This unusual result leads us to propose more fully exploringthe BDF.

In earlier efforts, the Handy group has identified a very mild andgeneral method for the synthesis of aurones (Hawkins et al., Tetrahedron2013, 69 (44), 9200-9204). (Scheme 2) Through this method, a wide rangeof over 80 new and non-natural aurone derivatives have been accessed inan efficient manner. Quite recently, a significant improvement upon thismethod has been discovered, whereby the use of microwave irradiation forheating results in comparable or even improved yields with reactiontimes of 30 minutes or less instead of 4-12 hours using conventionalheating. This dramatic reduction in time holds the potential to evenfurther accelerate the efforts to develop aurones into usefulanti-trypanosomal agents.

New generations of aurone-based derivatives can be designed andsynthesized using the three strategies outlined below.

Design and Synthesis of ADF Derivatives.

We have generated several anti-trypanosomal derivatives by focusing onADF substitutions. Some of the most promising aldehydes noted to dateare shown in Scheme 3. Additionally, dibromo and dichloro benzaldehydes(with one of the halogens in the ortho position) can continue to beexplored, as can commercially available p-cyanobenzaldehydes withadditional substitution. These aldehydes can be condensed with theparent unsubstituted benzofuranone and screened for activity.

In conclusion, the aldehyde-derived fragment of the aurone scaffoldprovides a rich and facile means of exploring a wide range of auroneanalogs. A number of these compounds exhibited significant levels ofactivity against both T. cruzi and T. brucei with good levels ofselectivity as well. When combined with their ease of synthesis, theymake very interesting lead compounds for further study and evaluation.

Design and Synthesis of BDF Derivatives.

We have also generated a compounds that focused on BDF substitutions. Anexample of the effects of simple structural modifications is shown inScheme 4.

The biological activity evaluations produced some surprising activityrelated to bromo, methyl, and hydroxyl substitutions on the BDF. Giventhe surprising success of methyl and bromo substitutions and thepotential for further elaboration, the benzofuranones shown in Scheme 5(all commercially available) will be condensed with the aldehydes in s 3and 7 to prepare a new library of aurones that will probe the BDFportion of the molecule. These studies will serve to probe the BDF interms of the tolerance of substitution at each of the 4 aromaticcarbons.

The generation of a further set of aurone analogs will involve theelaboration of the brominated BDFs that displayed good activity via arange of cross-coupling chemistry. This large family of versatilereactions has been little studied on halogenated aurones. (Scheme 6).

Suzuki and Sonogashira couplings have been studied so far in the Handygroup, while the Moreira group has reported Suzuki couplings andBuchwald-Hartwig aminations (Carrasco et al., Eur J Med Chem 2014,80:523-534). While these reactions could be used to introduce a numberof new aryl, alkenyl, alkynyl, alkyl, and amino groups (with this lastfamily being likely the most promising from a biological standpoint),there are many other copper-catalyzed coupling reactions that are worthyof study. These couplings are used to introduce nitro, cyano, azido, andeven ethers (oxy and thio) (Beletskaya et al., Coord Chem Rev 2004,248:2337-2364) (Scheme 6, dashed reaction arrow). While any of thesegroups are of potential interest, the nitro-substituted compounds areparticularly intriguing given the prior interest in nitro drugs for thetreatment of parasitic diseases (Peña et al., J. Sci Rep 2015, 5:8771).Given the generally, observed acceleration of coupling chemistry undermicrowave conditions, the use of the MARS reactor system will greatlyaid the parallel synthesis of these analogs.

The selection of where to incorporate these couplings will be directedin a two-fold manner. Sites in which neither bromination nor methylationare tolerated will be not be pursued further due to likely stericissues, while selected modifications will be explored on sites in whichthese initial modifications were tolerated. In addition, computationaldocking studies will be used to provide possible sites for enhancedbinding through targeted substitution and the biological results ofthese new compounds will be used to further fine-tune the model.

Design and Synthesis of Aza and Thioaurones.

A further area for exploration is compounds where the oxygen in thebenzofuranone ring is replaced with a nitrogen, a family commonly knownas azaaurones, or a sulfur, a family commonly known as thioaurones.Boumendjel reported a comparison of normal and azaaurones as potentialantimalarials (Souard et al., Bioorg. Med. Chem, 2010, 18 (15),5724-5731). In general, the aza series demonstrated greater potency(lower IC₅₀ values by a factor of 2), although relatively few directcomparisons were made. As their azaaurones were also oxygenated in theoxindole portion, the use of unsubstituted compounds as proposedrepresents a completely unexplored area. This nitrogen group is alsoexpected to improve the aqueous solubility properties of these compoundsrelative to simple aurones.

Aza and thioaurone compounds were synthesized based upon a modificationof that reported by Boumendjel (Souard et al., Bioorg. Med. Chem, 2010,18 (15), 5724-5731) as well as the earlier piperdine conditions for theaza series (Scheme 7). In our hands, the synthesis employing catalyticpiperdine often failed to afford good conversion and always affordedmixtures of the acetylated and deacetylated products, while the use ofaqueous potassium hydroxide in methanol was more reliable and onlyafforded the deacetylated product. In addition, the use of microwaveheating greatly reduced the reaction times from 3 hours underconventional heating to 30 minutes with microwave heating.

Two representative procedures for synthesis of azaaurones are asfollows.

1. KOH in Methanol Option

To a solution of 175 mg (1.00 mmol) of N-acetyl-3-hydroxyindole and therequired aldehyde (1 mmol) in 2 mL of methanol was added 2 mL of a 50%by weight solution of potassium hydroxide in methanol. This mixture washeated to 60 C in a GEM microwave reactor for 30 minutes. After cooling,the reaction was acidified with 1-N HCl and partitioned between ethylacetate and water. The organic layer was directly concentrated in vacuoand then purified via column chromatography on silica using ethylacetate/hexanes mixtures to afford the desired azaaurone product as asolid in 20-40% yield. These products displayed spectroscopic dataconsistent with the assigned structure.

2. Piperdine option. To a solution of 175 mg (1.00 mmol) ofN-acetyl-3-hydroxyindole and the required aldehyde (1 mmol) in 2 ml, oftoluene was added 2 drops of piperdine. The reaction was heated to 110 Cfor 30 minutes in a CEM microwave reactor and then cooled to roomtemperature. The resulting mixture was partitioned between water andethyl acetate. The organic layer was separated, concentrated in vacuo,and purified via column chromatography on silica using ethylacetate/hexanes mixtures to afford both the desired azaaurone product asa solid in 10-40% yield and the N-acetylated product in 3-20% yieldThese products displayed spectroscopic data consistent with the assignedstructure.

A representative procedure for synthesis of thioaurones is as follows.To a solution of 150 mg (1.00 mmol) of 1-thiobenzofuranone and therequired aldehyde (1 mmol) in 2 mL of methanol was added 2 mL of a 50%by weight solution of potassium hydroxide in methanol. This mixture washeated to 60 C in a CEM microwave reactor for 30 minutes. After cooling,the reaction was acidified with 1-N HCl and partitioned between ethylacetate and water. The organic layer was directly concentrated in vacuoand then purified via column chromatography on silica using ethylacetate/hexanes mixtures to afford the desired thioaurone product as asolid in 20-50% yield. These products displayed spectroscopic dataconsistent with the assigned structure.

Azaaurones and thioaurones synthesized according to the above proceduresare shown in Table 1 (Example II). They include the thioaurone,(Z)-2-((5-(hydroxymethyl)furan-2-yl)methylene)benzo[b]thiophen-3(2H)-one(TA2), as well as the following azaaurones:

-   (Z)-1-acetyl-2-(2-hydroxybenzylidene)indol in-3-one (AA3A)-   (Z)-1-acetyl-2-((5-(hydroxymethyl)furan-2-yl)methylene)indolin-3-one    (AA4A)-   (Z)-2-((1H-pyrrol-2-yl)methylene)indolin-3-one (AA5)-   (Z)-2-((1H-pyrrol-2-yl)methylene)-1-acetylindolin-3-one (AA5a)-   (Z)-2-benzylideneindolin-3-one (AA8)-   (Z)-2-(thiophen-2-ylmethylene)indolin-3-one (AA9)-   (Z)-2-(2-chlorobenzylidene)indolin-3-one (AA11)

Example IV. Evaluation of Aurone-Based Compounds for Anti-FungalActivity Introduction

The organism, Candida albicans, is a dimorphic fungus that is known tocause opportunistic infections of the oral cavity and genitalia inhumans. C. albicans is normally a commensal gut organism carried by alarge proportion of the population with no ill effects. The organisminfects host tissue by switching from the unicellular yeast form to amulticellular, invasive filamentous form. C. albicans causes a varietyof diseases under the collective term “candidiasis,” with C. albicans asthe most prevalent cause. The term candidiasis encompasses infectionsthat range from the superficial, such as oral thrush or the commonvaginal yeast infection, to the more serious infection candidemia, as isfound in immunocompromised patients with diseases such as AIDS, thoseundergoing chemotherapy treatments, or patients with implant surgeries(Kourkoumpetis et al., Virulence, 2010, 1:359-366).

Cryptococcus neoformans is a pervasive pathogenic yeast found in soiland other niches worldwide. In fact, 70% of urban children are infectedwith it by age 5 (Goldman et al., Pediatrics, 2001, 107:E66), and C.neoformans is becoming increasingly prevalent, especially in AIDSpatients. In most cases, infection does not present any immediateclinical symptoms; rather, it enters a chronic latent state in the hostthat may last for decades or even a lifetime. Active infection primarilyoccurs in individuals with compromised or damaged immune systems. Amongindividuals with compromised immune systems, acute infection or the“revival” of an existing infection can result in pneumonia andmeningitis. A rapid increase in the number of immune compromisedpatients has driven an exponential rise in clinical cases over the last30 years such that more than 1 million new infections and approximately600,000 deaths are attributed to C. neoformans each year (see, e.g.,Park et al., AIDS, 2009, 23:525-530; Rapp, et al., Pharmacotherapy,2004, 24:45-28S; Rabjohns et al., 2014, J. Biomolec. Screening, 19(2),270-277, epub Jul. 29, 2013; McClelland et al., 2007. Pathogenesis ofCryptococcus neoformans, in New Insights in Fungal Pathogenicity. Ed.Kavanagh, Springer; Centers for Disease Control and Prevention. (2014Dec. 2), C. neoformans Infection. Retrieved Jan. 15, 2015, fromhttp://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/index.html:Kauffman, Cryptococcosis. In: Goldman L, Schafer A I, eds. CecilMedicine. 24th ed, Philadelphia, Pa.: Saunders Elsevier; 2011:chap 344).

Immunocompromised patients in general have an increased risk ofcontracting a fungal infection. However, despite increased incidences ofimmunocompromised patients and invasive fungal disease, limitedantifungals are available and with a narrow range of targets: the cellwall, cell membrane, and DNA synthesis. Most of the drugs are alsoassociated with side effects or toxicity in their hosts and ofparticular concern is the emergence of resistance to many of thecommonly used antifungals for both C. albicans and C. neoformans(Perfect et al., Drug Resist Update, 1999, 2:259-269; Pfaller et al.,Clin Microbial Rev, 2007, 20:133-163; Brown et al., Sci Transl Med,2012, 4:165rv113; Li et al., Antimicrob Agents Chemother, 2015,59:5885-5891).

Commonly used antifungals can be grouped into classes based on theirsite of action: azoles, including, for example, fluconazole, andvorconazole, which inhibit the synthesis of ergosterol (the main fungalsterol); polyenes, including amphotericin B, which interact with fungalmembrane sterols physicochemically; 5-fluorocytosine (or5-fluorocytosine), which inhibits macromolecular synthesis; andechinocandins, including, for example, caspofungin, micafungin, andanidulafungin, which inhibit the synthesis of glucans found in fungalcell walls.

Amphotericin B is one of the most commonly utilized treatments forsevere fungal infections caused by fungal infections (Brajtburg, et al.,Antimicrob Agents Chemother, 1990, 34:183-188). Amphotericin B works bybinding ergosterol in fungal cell membranes and causing pore formation,leading to cell death. However, amphotericin B can be toxic to patients;common side effects in patients include kidney, liver, and heart damagedue to the similarity of lipids within both fungal and mammalian cellmembranes (Maddux et al., Drug Intell Clin Pharm, 1980, 14:177-181).

Azoles are antifungals that inhibit the biosynthesis of ergosterol,specifically via lanosterol 14-α-demethylase inhibition, which is theenzyme that converts lanosterol to ergosterol in yeasts (Georgopapadakouet al., Antimicrob Agents Chemother, 1987, 31:46-51; Sheehan et al.,Clin Microbiol Rev, 1999, 12:40-79). Azole resistance can occur due toalteration in drug target, the use of alternate sterol biosyntheticpathways, reduction of target enzyme, and/or overexpression of theantifungal drug target (Ghannoum et al., Clin Microbial Rev, 1999,12:501-517; Pfaller et al., Am J Med, 2012, 125:S3-13.).

The echinocandins, such as caspofungin and micafungin, inhibitbiosynthesis of 1,3-ß-D-glucan, an integral molecule in fungal cellwalls, via the disruption of 1,3-ß-D-glucan synthase. Without afunctioning synthase, yeasts are unable to maintain stable cell walls,leading to cell lysis. Resistance to this class of treatment occurs duein part to point mutations that prevent the inhibition of this enzyme(Pfaller et al., Am J Med, 2012, 125:S3-13).

Flucytosine (5FC), one of the oldest treatments for systemic fungalinfections, is an antifungal with no inherent antifungal activity.Converted to 5-fluorouracil within a fungal cell, 5FC inhibits fungalcell development by interfering with DNA and RNA synthesis. Resistancehas developed that prevent yeasts from taking up the drug and preventthe conversion of 5FC to 5-fluorouracil, a compound that prevents yeastsability to thrive (Pfaller et al., Am J Med, 2012, 125:S3-13).

Some antifungals have side effects more dangerous than the infectionitself. Of additional concern are the emergence of strains of C.albicans and C. neoformans resistant to many of the commonly usedantifungals. Resistance to the available treatments is rampant. Currenttherapies that control infections in patients with damaged immunesystems are associated with numerous problems, including drug toxicity,an inability to fully eliminate the infection, and the increasing drugresistance of C. neoformans strains. The toxicity, side effects andgrowing resistance to the currently existing antifungal agents create aneed for new and safer antifungal agents.

A group of biosynthetic precursor compounds related to aurones,flavonoids and chalconoids, possess antifungal activity (Cushnie, etal., Int J Antimicrob Agents, 2005, 26:343-356; Friedman et al., MolNutr Food Res, 2007, 51:116-134.) Through synthetic modification ofthese naturally occurring compounds, other antifungal compound classeshave been discovered, including 2,2-bisaminomethylated aurone analogues(Boumendjel, et al., Curr Med Chem, 2003, 10:2621-2630; Bandgar, et al.,Eur J Med Chem, 2010, 45:3223-3227).

Although aurones as a class of compounds have been previously suggestedto have antifungal activity (Haudecoeur et al., Curr Med Chem., 2012,19(18):2861-75; Bandgar et al., Eur J Med Chem., 2010, 45(7):3223-7;U.S. Pat. No. 6,307,070), neither Aurone 1009 nor Aurone 9051 has beenpreviously identified as having antifungal activity from among thepotentially vast number of synthetic aurone compounds.

Experimental

Selected members of the library of substituted aurones described inExample I were screened for antifungal activity versus Candida spp.,Cryptococcus spp., Saccharomyces cerevisiae and Trichophyton rubrum (afilamentous fungus). Selected substituted aurones screened forinhibitory effect against C. albicans are shown in Table 4A and FIG. 4.Several compounds displayed activity at 1.00 μM, with two having IC₅₀values below 20 μM for three species of Candida. One of the compoundstested here also exhibits anti-biofilm activity for mid-maturationgrowth. See, e.g., Sutton et al., Bioorg. Med. Chem, Lett., 15 Feb.2017, 27(4):901-903.

TABLE 4A Inhibitory effect of aurones against C. albicans C. albicansinhibition Sample (at ID Code Structure 100 μM) 2001

21.9% 2002

8.8% 2004

65.9% 2008

53.9% 2009

12.3% 2010

3.3% 2011

14.2% 2013

64.6% 2014

50.5% 2015

2.2% 2018

59.8% 2021

78.8% 2023

88.5% 2026

43.8% 1001

26.0% 1005

43.9% 1009

99.4% 5002

37.6% 5003

12.4% 5004

14.7% 5005

22.4% 5006

19.4% 6000

6.1% 7000

45.6% 7001

12.6% 8001

22.8% 8002

26.6% 2901

46.1% 2904

20.0% 2905

16.6% 2906

27.5% 2911

40.0% 2912

54.8% 9050

37.4% 9051

99.9% 9053

11.2% 9055

16.7% 9056

12.5% 9057

33.6% 9060

7.5% 9061

3.1% 9062

16.4% 9063

24.0% 9064

32.1% 9067

17.6% 9068

11.1% 9070

14.7% 9076

7.5% 9078

45.6% 9084

17.6% 9085

20.2% 9086

14.3% 9087

31.2% 9088

1.0% 9253

1.1%

Two main series of interest were identified within the set of structuresshown in Table 4A and are shown in Table 4B. The first was oxygenated(Table 4B, entries 1-8). In these cases, both the location and thedegree of oxygenation (and the presence of free phenols) were all foundto be important, with aurone 1009 proving to be the best. Ratherinterestingly, switching the relative position of the hydroxyl andmethoxy groups on the phenyl ring (1009 compared to 9053) or with twomethoxy groups (2011) or a reduction from two hydroxyl groups in thebenzofuranone portion to one hydroxyl group (2911) all resulted insignificant reductions in activity.

The second series focused on heteroaromatics, of which the pyridylsystem was optimal, with the 2-pyridyl compound 9051 being the best ofthe three pyridyl isomers. (Table 413, entries 10, 12, and 13) Otherheteroaromatics, including other nitrogen-containing ones were lessactive, with only the 2-pyrrolyl compound 2021 displaying even modestactivity. Thus, beyond the geometric considerations, it appears that thenucleophilicity of the nitrogen is of considerable importance as well inthis series.

TABLE 4B Screening Results for Selected Aurones against C. albicansPercent Compound Inhibition Entry^(a) Number Ar (at 100 μM)   1^(b) 1009

99.4 ± 4.7  2 9078

45.6 ± 2.6    3^(c) 2911

40.0 ± 6.5  4 2011

14.2 ± 6.1  5 9056

12.5 ± 0.4  6 9053

11.2 ± 2.2  7 9088

 1.0 ± 1.1  8 9055

16.7 ± 0.2   9^(b) 1001

26.0 ± 2.3 10 9051

99.9 ± 3.0 11 2021

78.8 ± 8.8 12 2008

53.9 ± 4.2 13 9253

 1.1 ± 0.1 14 2026

43.9 ± 2.3  15^(b) 2906

27.5 ± 2.2 ^(a)with unsubstituted benzofuranone except as noted.^(b)6,7-dihydroxybenzofuranone. ^(c)6-hydroxybenzofuranone.

Two of these compounds 9051 and 1009—displayed particularly high levelsof activity against C. albicans. These two aurones were investigated fortheir inhibitory activity against three different species of Candidas aswell as S. cerevisiae and T. rubrum (Table 5). Three different serotypesof C. neoformans, namely, serotype A (C. neoformans variety grubii),serotype D (C. neoformans variety neoformans) and serotype BIC (C.gattii) were tested as well (see Example V). For all Candida speciestested, IC₅₀ values below 20 μM were observed.

TABLE 5 IC₅₀ of each Aurone 1009 and Aurone 9051 for the yeasts C.albicans, C. glabrata, C. tropicalis, and S. cerevisiae and thefilamentous fungus T. rubrum Aurone C. albicans C. glabrata C.tropicalis S. cerevisiae T. rubrum

16 μM 11 μM 10 μM 26 μM 49 μM

18 μM 10 μM 12 μM 15 μM 50 μM

Aurone 1009 and Aurone 9051 demonstrate antifungal activity and arelikely to be of use as antifungal agents with potential applications inmedicine, agriculture, industry, and residential use.

Many microorganisms form structures called biofilms on a variety of bothliving and artificial surfaces. C. albicans is the most common fungus toform biofilms and differs physiologically from planktonic individuals ofthe same species sometimes becoming up to 1000 fold more resistant toantifungal treatment. C. albicans is known to form biofilms in manyinstances such as immunosuppressive therapy following transplantationprocedures and during the use of indwelling medical devices. When aCandida biofilm forms on an indwelling medical device such as acatheter, heart valve, artificial joint or a variety of other artificialsurfaces, candidiasis may occur. If infection occurs, treatment iseffective only after the artificial device is removed due to theantifungal resistance of the biofilm. In cases where the device is notremoved and an invasive candidiasis infection occurs, mortality rate hasbeen shown be as high as 40%.

C. albicans biofilms are divided into three stages, early phase (0-11h), intermediate (12-30 h), and maturation (31-72 h), with each havingdistinct properties. Inhibitory activity of aurones 1009 and 9051against intermediate C. albicans biofilms was investigated. Treatmentusing 1009 with the IC₅₀ concentration of 16 μM did not differsignificantly from the untreated control; however, the 1009 IC₉₉concentration of 100 μM shows was observed to cause complete disruptionof the biofilm, comparable to that observed with treatment by theantifungal amphotericin B, suggesting that aurones could haveapplication in the treatment of Candida biofilms. Aurone 9051 was notinhibitory to biofilms at concentrations tested.

Evaluation of Anti-Fungal Activity

A microdilution broth method (CLSI, Reference Method for Broth DilutionAntifungal Susceptibility Testing of Yeasts, Approved Standard-ThirdEdition, Clinical Laboratory Standards Institute, Wayne, Pa., 2008) wasfollowed with some modifications for the testing of potential antifungalcompounds Corning CellGro RPMI 1640 with glutamine, without bicarbonatebuffer, with phenol red (RPMI; Sigma-Aldrich) was used as the basemedium for testing. A 1 M solution of morpholinepropanesulfonic acid(MOPS; Sigma-Aldrich) was added to the RPMI to a final concentration of0.165 M for buffering (RPMI-MOPS). This medium was adjusted to a pH of7.0 with 10 M NaOH and filter sterilized.

C. albicans (ATCC strain 90028), C. glabrata (ATCC strain 66032), and C.tropicalis (ATCC strain 750) were cultured on potato dextrose agar(Sigma-Aldrich) plates, and incubated at 35° C. for 24 hours. Forantifungal testing, overnight colonies were adjusted to 1.5×10³cells/mL, hereafter referred to as the test inoculum.

C. neoformans (strain 1199s) was prepared following CLSI guidelines forthe organism (CLSI, Reference Method for Broth Dilution AntifungalSusceptibility Testing of Yeasts, Approved Standard-Third. Edition,Clinical Laboratory Standards Institute, Wayne, Pa., 2008).

PrestoBlue® (Life Technologies, Invitrogen, Carlsbad, Calif.), aresazurin-based cell viability reagent that, in metabolically activecells, is reduced from blue, non-fluorescent resazurin, to red,fluorescent resorufin, was used to assess inhibition of yeast growth bythe aurones. Reduction of the compound from resazurin to resorufin isproportional to the number of metabolically active cells and can bequantified by measuring relative fluorescence units (RFUs).

CoStar black-walled, clear bottom 96-well microtiter plates (FisherScientific, Waltham, Mass.) were seeded with 90 μl of test inoculum,treated with 10 μl of 1:2 dilutions of aurone in a range of 1 mM to 62.5μM to provide aurone having a final well concentration in a range of 100μM to 6.25 μM. Following addition of aurone, plates were then incubatedfor 24 hours at 35° C. PrestoBlue was added to a final concentration of10% in each well and fluorescence read at 560 nm excitation and 590 nmemission, per manufacturer instructions, using a SpectraMax M2espectrophotometer (Molecular Devices, LLC, Sunnyvale, Calif.).

To optimize the PrestoBlue reduction time, RFUs were collected at 15minute intervals for 4 hours using a reference antifungal. Thecalculated Z′, as described below, was compared to RFU readings and theoptimal PrestoBlue incubation time was determined to be 75 minutes.

Tissue culture treated eight-chambered slides (Fisher-Scientific) wereused for intermediate biofilm formation. C. albicans cells were added tothe chambers at a concentration of 5×10²-2.5×10³ cell s/mL in RPMI-MOPSmedia. 2 μg/mL Chamber slides were incubated for 24 h at 37° C. forbiofilm formation. After incubation, media was removed from the wells,wells were washed with RPMI-MOPS to removed planktonic cells, and mediareplaced with appropriate treatments, including medium alone(RPMI-MOPS), concentrations of aurones representing dilutions from theIC₉₉ to the IC₅₀, or the positive control treatment, 2 μg/mLamphotericin B, which has previously been shown to inhibit C. albicansbiofilm and is within the range for amphotericin B susceptibilitytesting (CLSI 2008, Reference method for broth dilution antifungalsusceptibility testing of yeasts. Approv Stand Ed. Generic, ClinicalLaboratory Standards Institute, Wayne, Pa.; Uppuluri et al., 2011,Antimicrob Agents Chemother, 55: 3591-3593). The slide was incubated anadditional 24 hours with appropriate treatments, and the slide was thenrinsed with PBS three times to remove planktonic cells (Samaranayake etal., J. Clin. Microbiol., 2005, 43:818-825). Calcofluor white(Sigma-Aldrich), a fluorescent dye that tightly binds to cellulose andchitin in the fungal cell wall, was used to qualitatively assess biofilmgrowth. Cells were visualized on an Olympus BX60 fluorescent microscopewith the laser and transmitted light on to illustrate biofilm density.

Statistical Analyses

Due to the nature of screening a large number of compounds, it isnecessary to determine the efficiency and quality of the assays on anindividual plate by plate basis. Zhang et al. (J Biomol Screen, 1991,4:67-73) developed a formula to assess the viability of an assay thataccount for the data variability present in screening that does not relyon test compound consistency, denoted as Z′. A calculated Z-factor (Z′)of 1.0 indicates an ideal assay, 1.0-0.5 an excellent assay, 0.5-0.0 anonviable assay and a Z′<0 indicates an assay that is impossible to use;all assays had a Z′ of greater than 0.8.

IC₅₀ determination was calculated using GraphPad Prism version 7.01 forWindows (GraphPad Software, Inc., La Jolla, Calif.) from the RFUs atdilution points. Calculations were made with a nonlinear regressionafter transforming the molar concentrations of the aurones into thelogarithmic form. Graphing was accomplished utilizing the same programfollowing a four parameter curve fitting sigmoidal plot of the data. Allaurones and controls were tested in triplicate and all testing wasrepeated in at least three independent experiments.

SUMMARY

Novel antifungals are in high demand as there is a growing resistance toantifungals currently in use. In particular, opportunistic fungalinfections caused by Candida spp. are on the rise with infections bythis genus accounting for the most severe fungal infections followingchemotherapy, implantation procedures, and in patients with HIV/ADS.

These results show that Aurone 1009 and Aurone 9051, as well as, to alesser extent, some of their derivatives, inhibit growth of fungalpathogens at low concentrations. Aurone 1009 and Aurone 9051 alsoinhibit and degrade biofilm growth by C. albicans. The effectiveness ofAurone 1009 and Aurone 9051 at low concentrations suggests that thesecompounds could be used to effectively treat infections of Candida spp.,C. neoformans, and other yeast species. More generally, activity againstS. cerevisiae, C. neoformans (see Example V) and multiple species ofCandida, as well as the filamentous fungus T. rubrum, suggests thataurones could serve as a potential broad-spectrum class of antifungalagents. The combination of the pyridyl ring with a hydroxylatedbenzofuranone may also represent a class of effective antifungal agents.

Example V. Characterization of Aurone 9051 as a Potential Drug CandidateAgainst Cryptococcus neoformans

Methods:

The A27-M2 CLSI standard micro-dilution method was used to screen anaurone library (MTSU Department of Chemistry) for inhibition ofCryptococcus neoformans (Cn). Each aurone was screened in triplicate.Compounds that showed inhibition were further tested to determine theminimum inhibitory concentration (MIC). Toxicity assays were conductedon human THP1 macrophages and L6 rat fibroblasts.

The inhibition of Cn strains and C. gattii strains by Aurone 9051 (alsoreferred to as “Aurone X”) was characterized in different medias(including RPMI 1640 (Standard), RPMI 1640+MOPS, Asparagine media,minimal media, or yeast extract peptone dextrose (YPD) media), againstother serotypes and strains, and at different cell concentrations.Synergy of the compounds with current drugs was characterized, and agrowth curve experiment was conducted.

Results:

Toxicity assays on human THP1 macrophages and L6 rat fibroblasts forindicated Aurone 9051 has low toxicity to mammalian cells.

Thirty-six extracts showed >90% inhibition of Cn at 100 μM. Aurone 9051was selected for further characterization based on its low MIC and itslow toxicity to THP1 macrophages and L6 rat fibroblasts (>100 μM). Asshown in Table 6, Aurone 9051 was found to inhibit Cn in RPMI+MOPS at12.5 μg/mL, in asparagine at 25 μg/mL, and in YPD at >200 μg/mL.Serotype A strains of C. neoformans, B18, B45, B58, and H99S had MICs of25 μg/ml, 25 μg/ml, 12.5 and 25 μg/ml, respectively Serotype D strainsof C. neoformans, 24067, JEC21, B3501, each had a MIC of 25 μg/ml. C.gattii strains of C. neoformans, R265 and R272, each had MICs of 25μg/mL. MIC was 25 μg/mL, 50 μg/mL, and 100 μg/mL for 10³, 10⁴, and 10⁵cells respectively.

Inhibition of Cn in different medias, at different cell concentrations,and with different serotypes was characterized (Table 6). Aurone 9051inhibits cidally at its WC and statically above its MIC (FIG. 7).

Aurone 9051 inhibits capsule growth, which is closely tied to the cellcycle (FIG. 6).

Aurone 9051 inhibits Cn growth early in its growth cycle (FIG. 5).

Aurone 9051 behaves additively with Amphotericin B and fluconazole andshowed no interaction with flucytosine (Table 7).

TABLE 6 Minimum Inhibitory Concentrations (MICs) of Aurone 9051 Sero-Inoculum MIC Yeast Strain type Media Type (CFU/ml) (μM) C. H99S A RPMI +MOPS 2 × 10³ 112 neoformans C. H99S A RPMI + MOPS 2 × 10⁴ 224 neoformansC. H99S A RPMI + MOPS 2 × 10⁵ 448 neoformans C. H99S A Asparagine 2 ×10³ 112 neoformans C. H99S A YPD 2 × 10³ >896 neoformans C. H99S AMinimal 2 × 10³ >4480 neoformans C. B18 A RPMI + MOPS 2 × 10³ 112neoformans (clinical) C B45 A RPMI + MOPS 2 × 10³ 112 neoformans(clinical) C. B58 A RPMI + MOPS 2 × 10³ 56 neoformans (clinical) C.24067 D RPMI + MOPS 2 × 10³ 112 neoformans (lab) C. JEC21 D RPMI + MOPS2 × 10³ 112 neoformans (lab) C. B3501 D RPMI + MOPS 2 × 10³ 112neoformans (lab) C. gattii R265 B/C RPMI + MOPS 2 × 10³ 112 C. gattiiR272 B/C RPMI + MOPS 2 × 10³ 112

TABLE 7 Antifungal interactions between Aurone 9051 and antifungalsagainst Cn. Fractional Inhibitory Antifungal - Aurone CombinationConcentration Index (FICi) Amphotericin B + Aurone 9051 0.625Fluconazole + Aurone 9051 0.75 Flucytosine + Aurone 9051 2 FICi valueswere calculated as follows: MIC of drug A in combination/MIC of drug Aalone + MIC of drug B in combination/MIC of drug B alone. FICi valueswere interpreted as follows: FICi < 0.5 synergy; 0.5 ≤ FICi ≤ 1additive; 1 < FICi < 4 indifferent; FICi > 4 antagonist.

Conclusions:

Aurone 9051 is a candidate for the treatment of C. neoformans and/or C.gattii infections. Drug interaction tests suggest Aurone 9051 may have amechanism of action similar to flucytosine, implying that its mechanismof action may include interfering with RNA/protein synthesis. SeeMuhammed, “Characterization of Aurone X as a Potential Drug CandidateAgainst Cryptococcus neoformans” Honors Thesis, Middle Tennessee StateUniversity, 2016-05, available athttp://jewlscholar.mtsu.edu/handle/mtsu/4853.

Example VI

Suppression of LPS-Induced NF-κB Activity in THP-1 and RAW 264.7 CellLines by the Synthetic Aurone, (Z)-2-((5-(hydroxymethyl) furan-2-yl)methylene) benzofuran-3(2H)-one

Introduction

Inflammation is a vitally important process, which can be triggered bystress, injury, or infection, and serves to protect the body fromharmful stimuli and pathogens and to facilitate the repair of damagedtissues. However, prolonged inflammation is associated with numerouschronic inflammatory and autoimmune disorders as well as cancer andneurodegenerative diseases. Chronic inflammation is known to beassociated with a wide variety of diseases and disorders; for exampleautoimmune disease such as rheumatoid arthritis (RA) and inflammatorybowel disease (IBD), obesity, diabetes, infectious diseases,inflammatory atherosclerosis, cancer, depression, heart disease, stroke,and Alzheimer's Disease. Inflammation can be chronic or acute; systemicor localized; autoimmune or associated with an infection caused by anexogenous agent. An autoimmune response is generally characterized as animmune response directed against a self-antigen. Inflammation caused byan exogenous agent, on the other hand, includes inflammation caused byan infectious agent or a pathogen such as a virus, bacteria, fungus,protist, plant, or other organism. Pathogenic bacteria known to induce achronic inflammatory response include Chlamydophila pneumoniae andPorphyromonas gingivalis(http://www.bumc.bu.edu/gencolablresearch/pathogen-induced-chronic-inflammatory-disorders/)

Inflammatory conditions and autoimmune diseases can be treated orprevented using immunomodulators or biologics, or both. Immunomodulatorsare compounds that weaken or modulate the activity of the immune system,which may in turn decrease the inflammatory response. Immunomodulatorsare used in organ transplantation to prevent rejection of the new organ,and to treat or manage autoimmune diseases such as rheumatoid arthritisand inflammatory bowel disease, which appears to be caused by anoveractive immune system. Exemplary immunomodulators includeazathioprine (available under the tradenames IMURAN and AZASAN),6-mercaptopurine (6-MP, available under the tradename PURINETHOL),cyclosporine A (available under the tradenames SANDIMMUNE and NEORAL),tacrolimus (available under the tradename PROGRAF), methotrexate(amethopterin, available under the tradenames MTX, RHEUMATREX, MEXATE,and TREXALL), hydroxychloroquine (available under the tradenamePLAQUENIL), leflunomide (available under the tradename ARAVA),sulfasalazine (available under the tradename AZULFIDINE), andminocycline (available under the tradename MINOCIN).

However immunomodulators are known to be accompanied by numerous sideeffects, including headache, nausea, vomiting, diarrhea, and malaise(general feeling of illness), pancreatitis (inflammation of thepancreas), bone marrow suppression, which may increase the risk ofinfection or serious bleeding, decreased kidney function, hepatitis,diabetes, increased cholesterol levels, sleep problems, mild tremor,high blood pressure, swollen gums, tingling of the fingers and feet,increased facial hair, and increased risk of lymphoma (a cancer of thelymphatic system), low white blood cell count, scarring of the liver andlung inflammation.

Whereas immunomodulators decrease the body's immune response, whichappears to be responsible causing the inflammation and damage associatedwith it, biologics are genetically engineered drugs that specific targetproteins or other molecules involved in the inflammatory process. Forexample, certain biologics block tumor necrosis factor-alpha, or TNF-α.TNF-α is an inflammatory cytokine that is present in elevated levels indiseases such as inflammatory bowel disease, and plays a central role inthe inflammatory response and damage to the GI tract that leads tosymptoms. Studies show that suppressing the production of importantpro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α)can effectively control inflammation (Brennan, Maini et al. 1995).Currently, biologics such as monoclonal antibodies and recombinantfusion proteins that target TNFα, are used to treat severe cases ofchronic inflammatory and autoimmune diseases (Thalayasingam and Isaacs2011). These biologics neutralize TNF-α's ability to cause inflammation.Biologics also have serious side effects, however, such as increasedrisk of mild to severe infection from the common cold to tuberculosis(TB) and hepatitis B, and increased risk of certain types of lymphoma,non-melanoma skin cancer, a lupus-like reaction, and exacerbation ofpre-existing heart failure. Three of the most widely used medications,infliximab (available under the tradename REMICADE), adalimumab(available under the tradename HUMIRA) and etanercept (available underthe tradename ENBREL) are antibodies (a type of “biologic” drug) thatact by binding to the cytokine tumor necrosis factor (TNF-α). However,the side effects associated with these medications, such as allergicreactions, increased risk of infections, malignancies, and risk ofstroke, can be severe. Unfortunately all three drugs come with a FDAblack box warning and have caused numerous problems and even death inpatients. Moreover, these drugs also only block the action of onecytokine (TNF-α). Other biologics in current use for management ofconditions such as rheumatoid arthritis or inflammatory bowel diseaseinclude tocilizumab (available under the tradename ACTEMRA) certolizumabpegol (available under the tradename CIMZIA), anakinra. (available underthe tradename KINERET), abatacept (available under the tradenameORENCIA) rituximab (available under the tradename RITUXAN) and golimumab(available under the tradename SIMPONI). There is therefore a clear needfor safe immunomodulatory agents for treating autoimmune diseases suchas rheumatoid arthritis and inflammatory bowel disease.

The severe side effects of long term use of the current treatments havedrawn attention to targeting intracellular signaling pathways such asthe NF-κB pathway (Barnes and Karin, 1997; Lewis and Manning, 1999). TheNF-κB (nuclear factor kappa B) pathway in particular is a key regulatorof the cellular response to stress and pathogens, controlling theexpressions of genes involved in proliferation, differentiation, cellsurvival, cell death, or pro-inflammatory response. The pathway consistsof a family of five transcription factors; Rel (c-Rel), RelA (p65),RelB, NF-κB1 (p50/p105), and NF-κB2 (p52/p100) (Verma et al., 1995)which share conserved homologous dimerization, transactivation, and DNAbinding domain (Ghosh et al., 1998). These proteins may form both homo-and heterodimers with the most prevalent activated form being p65 incomplex with either p50 or p52 (Schmitz and Baeuerle, 1991). The NF-κBpathway is mainly regulated by phosphorylation and ubiquination ofregulatory proteins such as inhibitor kappa B alpha (IκB α), whichmaintains canonical (i.e. p65-containing) transcription factors in aninactive state within the cytoplasm, and upstream kinases such asinhibitor kappa B kinase (IKK), which promote the proteasomaldegradation of IκB proteins and concomitant nuclear accumulation ofNF-κB transcription factors (Zandi and Karin, 1999).

Current treatments for immune-associated diseases and disorders, inaddition to causing serious side effects, block the action of only oneinflammatory cytokine, typically TNF-α. We have discovered a compound,however, more particularly an immunomodulatory aurone, that is able toblock the release of multiple cytokines. Without intending to be boundby theory, it is believed that the immunomodulatory aurone of theinvention acts at a step in a pathway (i.e., the NF-κB pathway) thatregulates cytokine production, rather than on the cytokine itself.

Summary

Suppressing cytokine responses has frequently been shown to havepromising therapeutic effects for many chronic inflammatory andautoimmune diseases. However, the severe side effects associated withthe long-term use of current treatments, such as allergic reactions andincreased risk of stroke, have focused attention towards the targetingof intracellular signaling mechanisms, such as NF-κB, that regulateinflammation.

Aurones are a sub-family of the flavonoids derived from plants with awide range of clinically-relevant activities including anti-cancer,anti-microbial, and anti-inflammatory activity. We have discovered anaurone that can act as an immunomodulator for treating or preventingimmune-based diseases or conditions, including autoimmune disease. Thefollowing example demonstrates that Aurone 1 in particular exhibitssignificant anti-inflammatory activity. Moreover, the immunomodulatoryaurone acts at a much earlier step in the cytokine pathway than thebiologics in current use, and blocks the release of multiple cytokines,a clear advantage over these biologics.

We synthesized a series of non-natural aurone derivatives andinvestigated their ability to suppress pro-inflammatory signaling inhuman monocyte (THP-1) and murine macrophage-like (RAW 267.4) celllines. One of these derivatives. (Z)-2-((5-(hydroxymethyl) furan-2-yl)methylene) benzofuran-3(2H)-one (aurone 1), was found to inhibitLPS-induced secretion of the pro-inflammatory cytokines, tumor-necrosisfactor α (TNFα), interleukin 1β (IL-1β), and IL-8 by THP-1 cells. Toinvestigate the mechanism, we probed the effect of aurone 1 onLPS-induced. MAPK and NF-κB signaling in both THP-1 and RAW264.7. Whileaurone 1 pre-treatment had no effect on the phosphorylation of ERK, JNK,or p38 MAPK, it strongly suppressed activation of IKK-β, as indicated byattenuation of Ser176/180 phosphorylation, resulting in decreasedphosphorylation of p65 (ser536) as well as phosphorylation (ser32) anddegradation of IκBα. Consistent with this, aurone 1 significantlyreduced LPS-stimulated nuclear translocation of p65-containing NF-κBtranscription factors and expression of an mCherry reporter of TNFα genetransactivation in RAW264.7 cells. Inhibition of TNFα expression at thetranscription level was also demonstrated in THP-1 by qRT-PCR. Inaddition to its effects on cytokine expression, aurone 1 pre-treatmentdecreased expression of iNOS, a bona fide NF-κB target gene and markerof macrophage M1 polarization, resulting in decreased NO production inRAW264.7 cells. Together, these data indicate that aurone 1 may have thepotential to function as a pharmacological agent for the treatment ofchronic inflammation disorders. See, e.g., Park et al., Int.Immunopharmacol., February 2017, 43:116-128.

Material and Methods Reagents

THP-1 (ATCC TIB 202) and RAW 264.7 (ATCC TIB 71) were purchased fromAmerican Type Culture Collection (Manassas, Va., USA). RAW 264.7 cellsstably expressing p65-EGFP fusion proteins from an endogenous p65promoter and also incorporating a destabilized mCherry reporter of TNF-αpromoter transactivation were a gift from Dr. Iain Fraser (NIH,Bethesda, Md., USA; (Sung, Li et al. 2014)). Lipopolysaccharide (LPS;Salmonella enterica serotype thyphimurium), dexamethasone,3-(4-methylphenylsulfonyl)-2-propenenitrile (Bay 11-7082), U0126(B5556), staurosporine, dimethyl sulfoxide (DMSO), phorbol 12-myristate13-acetate (PMA), protease inhibitors, phenylmethanesulfonyl fluoride(PMSF), ATP, and RPMI 1640 culture media were purchased fromSigma-Aldrich (St. Louis, Mo., USA). Dulbecco's Modified Eagle Medium(DMEM) was obtained from Corning Inc. (Corning, N.Y., USA). The fetalbovine serum (FBS), enhanced chemiluminescence luminol (ECL) substrate,penicillin/streptomycin, sodium pyrophosphate, SDS-PAGE gels, andnitrocellulose membranes were obtained from Fisher Scientific(Pittsburgh, Pa., USA). D-luciferin was purchased fromGold-Biotechnology (St. Louis, Mo., USA). Alamar blue was purchased fromLife Technologies (Grand Island, N.Y., USA). ELISA kits as well asassociated reagents were obtained from R&D Systems (Minneapolis, Minn.,USA). Bovine serum albumin (BSA) was obtained from EMD Millipore(Billerica, Mass., USA). L-glutamine and FBS for RAW 264.7 growth mediumwas obtained from GE Healthcare Life Sciences (Piscataway, N.J., USA).Cellomics NF-κB and BCA kits were purchased from Thermo Scientific(Waltham, Mass., USA). Antibodies for the Western blot analysis werepurchased from Cell Signaling Technology (Denver, Mass., USA).

Synthesis and Characterization of Aurone Derivatives

A synthetic scheme for various aurones is shown below. Reactionconditions used for the synthesis of selected aurone-derived compoundsfeaturing the deep eutectic solvent, choline chloride/urea, as both thereaction medium and catalyst for the reaction as well as the use ofmicrowave energy to greatly accelerate the reaction or the neutralalumina method (Varma and Varma 1992). The syntheses of compounds 1-9are reported in Example I.

-   -   A=Choline Chloride/Urea, 80 C, 12 hr    -   B=Neutral Alumina, CH₂Cl₂    -   C=Choline Chloride/Urea, microwave, 90 C. 30 min

Compound Ar Method Yield 1 2-(5-hydroxymethylfuryl) C 20% 22-(5-methylfuryl) C 61% 3 2-furyl A 54% 4 2-hydroxyphenyl C 54% 53-hydroxyphenyl C 84% 6 4-hydroxyphenyl B 34% 74-hydroxy-3-methoxyphenyl B 31% 8 2-hydroxy-3-methoxyphenyl B 18% 93-hydroxy-4-methoxyphenyl B 17%Table 8 shows the relationship between compound identifier 1-9, above,and aurone number in Table 1 (Example II).

TABLE 8 Aurone Identification Identifier Aurone 1 9067(Z)-2-(5-hydoxymethylfuran-2-yl)methylene)benzofuran- 3(2H)-one 2 9251(Z)-2-(5-methylfuran-2-yl)methylene)benzofuran- 3(2H)-one 3 2023(Z)-2-(furan-2-yl)methylene)benzofuran-3(2H)-one 4 9088(Z)-2-(2-hydroxybenzylidene)benzofuran-3(2H)-one 5 9252(Z)-2-(3-hydroxybenzylidene)benzofuran-3(2H)-one 6 9068(Z)-2-(4-hydroxybenzylidene)benzofuran-3(2H)-one 7 9053(Z)-2-(4-hydroxy-3-methoxybenzylidene)benzofuran- 3(2H)-one 8 9055(Z)-2-(2-hydroxy-3-methoxybenzylidene)benzofuran- 3(2H)-one 9 9078(Z)-2-(3-hydroxy-4-methoxybenzylidene)benzofuran- 3(2H)-one

Maintenance and Differentiation of the THP-1 and RAW 264.7 Cell Line.

THP-1 cells were maintained in RPMI 1640 medium supplemented with 10%heat-inactivated FBS and 1% penicillin/streptomycin (complete culturemedium) at 37° C. with 5% CO₂ supplemented. Cell concentrations wereadjusted to desired concentrations for each experiment by centrifugationat 500×g for 5 min and resuspended in complete culture medium with 100nM of PMA. Cell concentration was adjusted to 5×10⁵ cells/ml for allassays with the exception of 2.5×10⁵ cells/ml were used for NF-κBnuclear translocation assay. Cells were seeded onto 96-, 24-, or 12-wellplates and incubated for 48 to 72 h to allow for differentiation. Cellswere washed with serum-free RPMI 1640 medium before each experiment toremove undifferentiated cells. RAW 264.7 cells were maintained inDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%heat-inactivated FBS, 200 mM L-glutamine, 1% penicillin/streptomycin,and 50 μg/ml gentamicin (complete culture medium) at 37° C. in ahumidified atmosphere supplemented with 5% CO₂. Cells were seeded intotissue culture plates at 10% confluence and grown to 80% confluencewithin 72 h. For live cell microscopy, 1×10⁵ cells/ml were seeded into35 mm glass-bottom (MatTek) dishes 24 h prior to imaging. For luciferaseassays, 1.2×10⁵ cells/well were seeded into 24-well tissueculture-treated plates.

Alamar Blue Cell Viability Assay.

Differentiated THP-1 and RAW264.7 cells were treated with a range ofconcentrations of aurone derivatives for 1 h and stimulated with 20ng/ml of LPS for 4 h. Following treatment, the relative cell viabilitywas measured by Alamar Blue assay. For Alamar Blue assays, supernatantswere replaced with culture medium containing 1× Alamar Blue reagent andincubated overnight. Cell viability was assessed by measuring relativefluorescent units (RFU) on the SpectraMax M2e microplate reader(Molecular Devices Inc., Sunnyvale, Calif., USA) at Ex 560 nm and Em 590nm. The results were expressed as a percentage relative to LPS alonecontrol cells. The effects of the vehicle control, DMSO, on cellviability were also assessed.

Assessment or Cytokine Response by ELISA.

Differentiated THP-1 cells were pretreated with a range ofconcentrations of aurone derivatives or 1 μM of dexamethasone for 1 hand stimulated with 20 ng/ml of LPS for 4 h. Dexamethasone is asynthetic glucocorticoid that suppresses LPS-induced pro-inflammatorycytokine expression and was used as a control (Abraham, Lawrence et al.2006). Supernatants were collected for human cytokine ELISAs and themanufacturer's protocol was followed to assess the cytokine response.Cells remaining after the supernatant collection were tested forrelative viability by Alamar Blue cytotoxicity assay as previouslydescribed.

Indirect immunofluorescence for NF-κB Nuclear Translocation.

Differentiated THP-1 cells were pretreated with 50 μM of aurone 1 or 10μM of Bay 11-7082 for 1 h and stimulated with 1 μg/ml of LPS for 30 min.Bay 11-7082 is a compound that inhibits LPS-induced activation ofIKKα/β, thereby suppressing downstream IκBα phosphorylation/degradationand p65 nuclear translocation and was used as a positive control(Catalan, Fernandez-Castillejo et al. 2012). Treated cells were fixed,permeabilized, blocked, and stained with p65 (NF-κB) primary antibody,Dylight 488 conjugated secondary antibody, and Hoechst 33342 dye,sequentially. The Hoechst and DyLight fluorophores detect changes innuclear morphology (blue fluorescence) and NF-κB distribution (greenfluorescence), respectively. Nuclear Translocation Bioapplicationsoftware on the Arrayscan VTI reader was used for image acquisition anddata analysis (Thermo Fisher Scientific, Waltham, Mass., USA). For eachwell, at least 400 cells were automatically acquired and analyzed. Thetranslocation index was calculated by measuring the average intensitydifference of NF-κB between the identified cytoplasmic region andnuclear region.

Live Cell Imaging of RAW 264.7 Cells.

RAW 264.7 cells were pretreated with 50 μM of aurone 1 for 1 h,stimulated with 20 ng/ml LPS, and imaged every 3 min over 5 h using aNikon Ti-Eclipse wide-field microscope (Nikon, USA), equipped with aCoolSNAP Myo camera (Photometrics, AZ, USA), computer-controlled stage,and full environmental enclosure (InVivo Scientific, MO, USA). Cellswere maintained at 37° C. with 5% CO2 in a humidified atmosphere duringimaging. EGFP and mCherry fluorescence were imaged using FITC and Cy3filters, respectively. Nikon Elements Software (Nikon. USA) was used formicroscope control and image capture. Post-acquisition, images wereanalyzed using Fiji (Schindelin, Arganda-Carreras et al. 2012). Imageswere background subtracted and cytoplasmic:nuclear p65-EGFP and wholecell mCherry fluorescence were quantified for individual cells at eachtime point.

Luciferase Assay.

RAW 264.7 cells were transfected with 1.5 μg endotoxin free pNF-κB-Luc(Stratagene, UK) 24 h prior to being pretreated with 50 μM of aurone 1for 1 h, and then stimulated with 20 ng/mL LPS for 6 h. Cells were lysedin 250 μl/well of luminometry lysis buffer [25 mM Tris-phosphate,1%_((w/v)) BSA, 0.025%_((w/v)) dithiothreitol (DTT), 1% Triton X-100,15%_((v/v)) glycerol, 0.1 mM EDTA, 8 mM MgCl₂, 1× protease inhibitorcocktail, and 1 mM phenylmethylsulfonyl fluoride] and incubated on ashaking table at 200 rpm for 15 min. A volume of 10 μl of 25 mM ATP wasadded to each well, and the samples were transferred in duplicates of100 μl to an opaque-white 96-well plate. A volume of 20 μl of 10 mMsodium pyrophosphate was added to each well prior to the addition of 100μl of 2 mM D-luciferin. Luminescence was quantified using a SpectraMaxM5 plate reader using SoftMax Pro 6.3 software (Molecular Devices,Sunnyvale, Calif., USA).

Western Blot Analysis.

Differentiated THP-1 or RAW 264.7 cells were pretreated with a range ofconcentrations of aurone 1, 10 μM of U0126, or 10 μM of Bay 11-7082 for1 h and stimulated with 1 μg/ml of LPS for the indicated times. U0126 isa compound that inhibits MEK1/2 and was used as positive control for ERKphosphorylation inhibition (Hotokezaka, Sakai et al. 2002). Cells werelysed with radio-immunoprecipitation assay (RIPA) lysis buffer thatcontained a protease and phosphatase inhibitor cocktail. Cell lysateswere then tested for protein concentration using a BCA protein assay anddiluted with RIPA lysis buffer to normalize protein concentration in allsamples. Lysates were mixed with sample loading buffer containingbromophenol blue, glycerol, SDS, and 2-mercaptoethanol. The separatedproteins were then transferred onto a nitrocellulose membrane andblocked with 5% BSA in IX Tris-buffered saline with 0.1% Tween-20 for 1h. The blots were incubated with primary antibodies at 4° C. overnightfollowed by incubation with HRP-conjugated secondary antibodies for 2 hat 22° C. The membranes were developed by addition of ECL substrate andimages were collected with a ChemiDoc XRS+ system chemiluminescenceimager (Bio-Rad, Hercules, Calif., USA). Western blot band intensity wasquantified using Image Lab software (Bio-Rad, Hercules, Calif., USA).

Antibodies.

Antibodies used for Western blot analysis were as follows: Actin (A2066,Sigma; SC-1616, Santa Cruz Biotechnology, Dallas, Tex., USA), and iNOS(D6136S), IKK β (D3006), phosphorylated IKK α/β (Ser176/180; 16A6), IκBα (L35A5), phosphorylated IκB α (Ser32; 14D4), p65 (D14E12),phosphorylated p65 (Ser536; 93H1), SAPK/JNK (9252), phosphorylatedSAPK/JNK (Thr183/Tyr204; 81E11), ERK1/2 (p44/42; 137F5), phosphorylatedERK1/2 (Thr202/Tyr204; D13.14.4E), p38 (D13E1), phosphorylated p38(Thr180/Tyr182; D3F9), anti-rabbit IgG, HRP-linked (7074), andanti-mouse IgG, HRP-linked (7076) were all purchased from Cell SignalingTechnology (Denver, Mass., USA).

Quantitative RT-PCR.

Transcription of TNF-α (NM 000594) and beta-2-microglobin, 132M (NM004048), in 2.5×10⁵ PMA-differentiated THP1 cells was investigated incells without treatment, stimulated with 20 μg/ml Salmonella LPS, orpretreated for 1 hour with 1 μM dexamethasone or 80 μM aurone 1 followedby 4 hours of incubation with 20 ng/ml LPS. Total RNA was purified after4 hours of LPS stimulation using the Maxwell 16 LEV simplyRNA Tissue Kit(Promega, WI, USA) per manufacturer's instructions (Jeffries, Kiss etal. 2014). Total RNA concentrations and the 260/280 nm ratios of eachRNA sample were assessed using a NanoDrop 2000 UV-Vis spectrophotometer(Thermo Fisher Scientific, MA, USA). SYBR® FAST One-step qRT-PCR (KAPABiosystems, MA, USA) was used to examine gene transcription afterreaction optimization.

QuantiTect® Primer Assay TNF (QT01079561) and B2M (Q300088935) primer(Qiagen, CA, USA) optimization studies, including melt curve analyses toconfirm primer specificity and efficiency, were conducted to findappropriate running conditions (Fajardy, Moitrot et al. 2009). Briefly,the optimized conditions were 1 ng of total RNA added to 20 μl ofreaction including 150 nM of forward and reverse primers. Bio-Rad CDCConnect real-time PCR detection system (Bio-Rad, CA, USA) cyclingconditions, were as follows: 42° C. for 10 min, 95° C. for 3 minfollowed by 40 cycles of 95° C. for 3 s and 60° C. for 20 s. Samplescontaining no cDNA template or no primers were used as negative templateor negative reverse-transcription controls, respectively. HumanXpressRef Universal Total RNA (Qiagen, CA, USA) was used as a positivecontrol. Using optimized conditions, triplicate samples were assayedfrom three biological replicates (n=9). The fold change was calculatedby using 2^(−ΔΔct) normalized to LPS alone (Schmittgen and Livak 2008).

NO Assay.

Nitrite concentration in culture media of cells exposed to the indicatedtreatments was determined as an estimate of NO production with Griessreagent using a nitrate/nitrite colorimetric assay kit (Cayman ChemicalCompany, USA) according to the manufacturer's protocol.

Statistical Analysis.

All experiments were conducted at least three times independently.Statistical significance was determined using GraphPad Prism 6(GraphPad, La Jolla, Calif., USA). Numeric values of treated groups werecompared to the control group and results were expressed as mean±SEM.Statistical significance was analyzed using one-way analysis of variancefollowed by the Sidak test (GraphPad Prism). A value of p<0.05 was setfor significance.

Results

Synthesis and Characterization of Aurone Derivatives.

The synthetic aurones were all prepared via the standard condensation ofcoumaranone with the appropriate aldehyde under three different sets ofconditions, as shown above. Four were prepared in modest yield using theconditions reported by Varma and Varma (1992) (neutral alumina inmethylene chloride) (aurones 6-9). Aurone 3 was prepared using the verymild conditions reported by Hawkins and Handy (2013) using cholinechloride/urea as the solvent and catalyst. Finally, four were preparedby the combination of the choline chloride/urea reaction conditions withmicrowave heating (aurones 1, 2, 4, and 5). This method combines verymild reaction conditions with a short reaction time, and enabled aurone1 to be prepared in 20% yield as compared to 8% using conventionalheating and a 2% yield under the Varma and Varma (1992) conditions.Another key modification of the reaction conditions reported earlier isthe observation that purification of the crude reaction product can bereadily performed by simple trituration with ether instead of columnchromatography. This change has generally afforded much-improved yieldsand certainly decreases the time required to prepare new auronederivatives. Aurones 1-9 are shown in Table 9.

TABLE 9 Aurone derivatives Aurone # Structure IUPAC Name 1 (Compound9067)

(Z)-2-(5-hydroxymethylfuran-2- yl)methylene)benzofuran-3(2H)- one 2(Compound 9251)

(Z)-2-(5-methylfuran-2- yl)methylene)benzofuran-3(2H)- one 3 (Compound2023)

(Z)-2-(furan-2- yl)methylene)benzofuran-3(2H)- one 4 (Compound 9088)

(Z)-2-(2- hydroxybenzylidene)benzofuran- 3(2H)-one 5 (Compound 9252)

(Z)-2-(3- hydroxybenzylidene)benzofuran- 3(2H)-one 6 (Compound 9068)

(Z)-2-(4- hydroxybenzylidene)benzofuran- 3(2H)-one 7 (Compound 9053)

(Z)-2-(4-hydroxy-3- methoxybenzylidene)benzofuran- 3(2H)-one 8 (Compound9055)

(Z)-2-(2-hydroxy-3- methoxybenzylidene)benzofuran- 3(2H)-one 9 (Compound9078)

(Z)-2-(3-hydroxy-4- methoxybenzylidene)benzofuran- 3(2H)-one

Cytotoxicity of Aurone Derivatives in THP-1 and RAW 264.7 Cells.

In order to determine the effect of aurones on inflammatory signaling byinnate immune cells, we first assayed the toxicity of our auronecompounds on THP-1 cells in combination with LPS. The viability test wasperformed for every supernatant sample collection used for the cytokineresponse assay to show that suppression of cytokine response was not dueto cell death. Cells pretreated with 20, 40, 80 μM of aurone 1 with LPSfor 4 h had no effect on cell viability, as measured using Alamar Blueassay (FIG. 8A). However, treatment with aurone 1 at 100 μM with LPSresulted in less than 90% viability, therefore 80 μM was selected as themaximum concentration for subsequent experiments in THP-1 cells.Corresponding assays were also performed in RAW 264.7 cells withconcentrations up to and including 100 μM of aurone 1 with LPSexhibiting no effect on viability at 4 h post treatment (FIG. 8B).Furthermore, we also found that RAW 264.7 cells could be incubated withthis higher dose of aurone 1 for at least 24 h without apparent toxicity(FIG. 8C). Table 10 shows the maximum concentrations of other auronederivatives that were non-toxic to THP-1 cells. The vehicle control,DMSO, was also tested for toxicity and showed no effect on viability ineither cell line (data not shown).

TABLE 10 The average percent viability and % TNF-α inhibition of theTHP-1 cells treated with various concentrations of aurone derivativeswith EPS, Data are expressed as mean ± SD of triplicates for eachexperiment. Average % Concentration Viability Average % TNF-α Compounds(μM) (4 h with LPS) Inhibition 1 20 96.0 ± 0.6 25.3 ± 6.1 Compound 906740 96.8 ± 0.9  51.8 ± 13.5 80 103.1 ± 0.2  93.8 ± 0.7 2 25 99.4 ± 9.3 NoInhibitions Compound 9251 50 118.3 ± 1.3  100 108.9 ± 2.0  3 12.5 104.3± 2.2  No Inhibitions Compound 2023 25 102.1 ± 0.8  50 92.2 ± 0.9 4 2099.4 ± 0.5 No Inhibition Compound 9088 40 98.8 ± 1.6 15.4 ± 5.3 5 2094.6 ± 1.2 No Inhibitions Compound 9252 25 92.9 ± 1.4 40 90.9 ± 0.6 6 2099.8 ± 1.5 No Inhibitions Compound 9068 40 98.5 ± 0.1 7 20 97.0 ± 0.3 NoInhibition Compound 9053 40 99.6 ± 0.1 10.4 ± 4.7 8 20 101.3 ± 2.6  NoInhibitions Compound 9055 40 100.9 ± 0.3  9 20 101.8 ± 2.1  NoInhibition Compound 9078 40 100.9 ± 1.3  14.9 ± 7.2

Effects of Aurone 1 on TNF-α, IL-1β, and IL-8 Response in LPS-StimulatedTHP-1 Cells.

We next investigated the effects of aurone derivatives on expression ofthe inflammatory cytokine, TNFα, by ITS-stimulated THP-1 macrophagesusing ELISA. ITS is recognized by the pattern recognition receptor, TLR4with CD14 and other associating proteins on the surface of the membrane,triggering MyD88-dependent activation of a number of transcriptionfactors that regulate the expression of TNFα and other inflammatoryregulators (e.g. NF-κB and AP-1). While aurone 1 suppressed TNFαexpression by 93.8% (±0.7; FIG. 9A), aurones 4, 7, and 9 at 40 μM causedonly modest inhibition (< than 15%), and all others failed to show theeffect (Table 10). Therefore, only aurone 1 was selected for furtherstudy. In addition to suppressing TNFα secretion, aurone 1 also reducedexpression of IL-1β, and IL-8 in THP-1 cells by 98% and 71% respectively(FIG. 9B-D), in concentration-dependent manner. The fold changes incytokines are shown in FIG. 9D and fold changes of treated groups arerelative to the ITS-only group. DMSO, which was used as a vehicle foraurone 1, did not affect cytokine responses in LPS-treated THP-1 cells(data not shown).

Aurone 1 Inhibits TNFα Production at the Transcriptional Level.

As TNF, IL1B, and IL8 are bona fide NF-κB responsive genes (Collart,Baeuerle et al. 1990, Shakhov, Collart et al, 1990, Hiscott, Marois etal. 1993, Kunsch and Rosen 1993, Kang, Kim et al. 2007), we hypothesizedthat the reduction in ITS-induced cytokine expression in response toaurone 1 (i) occurred at the transcriptional level and (ii) was achievedthrough inhibition of canonical NF-κB activity. To test these hypotheseswe measured LPS-induced TNFα mRNA levels in aurone 1 pre-treated THP-1cells by qRT-PCR and NF-κB dependent transcription in RAW 264.7 cellsusing a luciferase reporter assay. Here, we found that pre-treated with80 μM aurone-1 reduced TNFα mRNA levels in differentiated THP-1 cells by49% (p≤0.0001) (FIG. 12A). To test the effects of aurone 1 onNF-κB-dependent transcription, RAW264.7 cells were transientlytransfected with the pNF-κB-Luc reporter construct, which contains thefirefly luciferase gene under the control of a promoter containing 5tandem consensus κB sites. Here, pre-treatment with aurone 1 was foundto significantly inhibit LPS-induced luciferase expression in thesecells (FIG. 12B). Together, these data strongly suggested that theeffects of aurone 1 on cytokine expression were a consequence of NF-κBinhibition.

Aurone 1 Inhibits LPS-Induced Nuclear Translocation of p65 in Human andMarine Macrophages.

Having shown that aurone 1 could block NE-κB dependent transcription, wetested whether this was due to the inhibition of nuclear translocationof the canonical NF-κB transcription factor, p65 (Rel A). Usingimmunofluorescent staining of THP-1 cells challenged with LPS for 30min, we found that pre-treatment with 50 aurone 1 was capable ofblocking cytoplasmic-to-nuclear translocation of p65. This wascomparable to the effects of the IKK inhibitor, Bay 11-7082, whichblocked p65 translocation at a dose of 10 μM (FIG. 10). To test whetheraurone 1 had similar effects in murine macrophages and to investigatehow it might alter the kinetics of the NF-κB response to LP'S, weemployed a previously described NF-κB dual reporter RAW 264.7 murinemacrophage cell line (Sung, Li et al. 2014). The reporter cell line,which stably expressed an EGFP fusion of p65 (p65-EGFP), alsoincorporates an exogenous reporter of TNFα gene transactivation based onthe core murine TNF promoter (−1229 to −27) regulating the expression ofthe red fluorescent protein, mCherry. Using live cell microscopy ofthese dual reporter cells, we found that nuclear translocation ofp65-EGFP post-LPS treatment was diminished in cells pretreated withaurone 1 and occurred slightly later (FIG. 11A-D). The reduction innuclear p65 levels, expressed as the ratio of nuclear to cytoplasmicp65-EGFP fluorescence, was found to be statistically significant (FIG.11E). Consistent with these data and our qRT-PCR analysis of TNF genetranscription in THP-1 cells (FIG. 12A), the reduction in nuclear p65resulted in smaller fold-change in the expression of mCherry in themurine dual reporter cells (FIG. 6A, F-H). This was also found to besignificant (FIG. 11.1).

Aurone 1 Inhibits IKKβ, IκBα, and p65 Phosphorylation.

Since p65 nuclear translocation was attenuated by aurone 1 in both THP-1and RAW 264.7 cells, we measured US-induced changes in thephosphorylation of the critical upstream regulators of p65, IKKβ andIκBα, as well as phosphorylation of p65 itself. Phosphorylation of IKKβat ser176/180 is required for kinase activity and phosphorylation of itssubstrate, IκBα, at ser32/36, which stimulates its ubiquitination andproteasomal degradation (Yang, Tang et al. 2003, Barisic, Schmidt et al.2010). Phosphorylation of p65 at ser536 has also been associated withincreased transcriptional activity (Buss, Dorrie et al. 2004, Hoberg,Popko et al. 2006). Like the inhibitor of IKK activation, Bay 11-7082,aurone 1 was found to suppress the phosphorylation of all three proteinsin both THP-1 (FIGS. 13A and C-E) and RAW 264.7 cells (FIGS. 13B andF-H). In all cases, these effects were found to be dose-dependent.However, differences were observed in the magnitude of the responsebetween the two cell lines with aurone 1 appearing to have a greatereffect on IKK phosphorylation in THP-1 cells than in RAW264.7 cells(FIG. 13C+F). Despite this, aurone 1 strongly suppressed IκBα and p65phosphorylation direct targets of IKKβ even at 25 μM doses in RAW 264.7cells (FIG. 13G+H).

Aurone 1 does not Significantly Affect MAPK Phosphorylation.

In addition to stimulating NF-κB activity, LPS also promotes thephosphorylation of ERK, JNK, and p38, leading to the activation of AP-1transcription factors. As AP-1 also regulates the expression of TNFα andother pro-inflammatory cytokines at the transcriptional level (Liu,Sidiropoulos et al. 2000), we hypothesized that inhibition of MAPKs maycontribute to the decreased expression of TNFα seen in aurone 1 treatedcells. To test this possibility, we measured the phosphorylation ofMAPKs in both THP-1 and RAW264.7 cells (FIG. 14A-H). We found thataurone 1 had no statistically significant effect on the phosphorylationof ERK (FIGS. 14A+B and D+G), JNK (FIGS. 14A+B and C+F), or p38 (FIGS.14A+B and E+H) across both cell lines tested, strongly indicating thataurone 1 was not affecting cytokine expression via altered NF-κB but notMAPK activity.

Aurone 1 Decreases iNOS Expression and NO Production in RAW264.7 Cells.

As well as pro-inflammatory cytokines, LPS is known to induce a plethoraof other NF-κB-responsive genes in macrophages (Sharif, Bolshakov et al.2007), including NOS2 (Xie, Kashiwabara et al. 1994), which encodesiNOS, a well-established marker of M1 macrophage polarization. To testwhether this was also affected by aurone 1, we measured iNOS expressionin LPS-stimulated RAW264.7 cells with and without aurone 1 pre-treatmentby Western blotting. Like TNFα, we found that iNOS production wasreduced by aurone 1 in a dose-dependent manner, with 50 and 100 μMproducing a 31.7 and 57.5% reduction in iNOS levels, respectively (FIG.15A). Furthermore, production of NO, the product of iNOS, wassignificantly reduced in the supernatants of aurone 1 pre-treated cells(FIG. 15B). Together, these data suggest that aurone 1 can at leastpartially block M1 polarization of macrophages.

Discussion and Conclusion

Inflammation is triggered by multiple factors including stress, trauma,and infection. While under normal circumstances, this process isself-limiting, resulting in an appropriate and transient inflammatoryresponse, in the disease state inflammation can becomeself-perpetuating. Pro-inflammatory regulators inducing the productionof further pro-inflammatory regulators in a positive-feedback loop canresult in destructive chronic inflammation and is associated with thedevelopment of degenerative diseases such as cardiac disease, cancer,neurodegenerative disorders, stroke, and diabetes (Ridker, Cushman etal. 1997, Bastard, Maachi et al. 2006, Kundu and Surh 2008, Amor,Puentes et al. 2010). Currently available preventive therapy for chronicinflammatory and autoimmune diseases typically targets the cytokineresponse with anti-TNF therapy being clinically demonstrated as the mosteffective approach to control inflammation (Postal and Appenzeller2011).

The downside of TNF blockers is that they can cause severe side effectssuch as allergic reactions, increased risk of infections, malignancies,and stroke, and are thus limited to severe inflammatory diseases such asrheumatoid arthritis and ankylosing spondylitis (Bjamason, Hayllar etal. 1993, Bongartz, Sutton et al. 2006, Bezalel, Asher et al. 2012,Diamantopoulos 2013). Thus targeting the signaling pathways thatregulate cytokine expression—including NF-κB—with small molecule-basedinhibitors is seen as an attractive alternative.

The present study describes the characterization of a novel syntheticaurone, which is capable of suppressing LPS-induced expression ofinflammatory cytokines and iNOS expression by blocking the activation ofthe canonical NF-κB pathway. The effects of this compound were alsofound to be highly consistent between species with the phosphorylationand nuclear translocation of p65 proteins found to be stronglysuppressed in both human THP-1 and murine RAW264.7 cell lines. Theutilization of live cell imaging, which has frequently been used by ourand other groups to study NF-κB signaling (Nelson, Ihekwaba et al. 2004,Ashall, Horton et al. 2009, Lee, Walker et al. 2014, Sung, Li et al.2014, Hayes, Sircy et al. 2016), allowed us to probe both the impact ofthe aurone on p65 translocation and the downstream transcriptionalconsequences in individual cells. Here, we found that decreased nuclearaccumulation of p65 resulted in a corresponding loss of expression ofthe reporter gene, mCherry, which was expressed from a portion of themurine TNF promoter, which incorporates KB sites. Although all fourtarget genes assayed in this study are co-regulated by MAPK, which isalso induced by LPS/TLR4 signaling, we found that aurone 1 had no effecton ERK, JNK1, and p38 MAPK. We therefore can conclude that the primarymechanism by which aurone 1 suppresses the expression of these genes isvia NF-κB. However, the identity of the specific molecular target ofaurone 1 remains an open question and is the subject of on-goingstudies. Based on its contrasting effects on NF-κB and MAPK signaling,we speculate that aurone 1 may directly affect the activity of the IKKcomplex itself. Alternatively, aurone 1 may have a differential effecton a common upstream regulator of the two pathways. One possiblecandidate might be transforming growth factor β-activated kinase 1(TAK1), a divergence point for LPS/TLR4-induced NE-κB and MAPKsignaling. While the regulation of TAK1 is not yet fully understood,recent data has suggested that the ability of this protein to induce IKKand MAPK activity is somewhat separable with certain modifications, suchas ubiquitination of Lys158 being required for both IKK and MAPKinduction (Fan, Yu et al. 2010, Fan, Yu et al. 2011), whilephosphorylation within the activation loop of TAK1 is dispensable forIKK activation (Chen, Hsu et al. 2015). We also cannot rule out thepossibility that aurone 1 affects other kinases that influence NF-κBactivity in LPS-stimulated macrophages. Integrin-linked kinase (ILK),for example, has also been shown to phosphorylate p65 at ser536 inRAW264.7 cells with knockdown or inhibition of ILK decreasing TNFαexpression in these cells (Ahmed, Sarvestani et al. 2014).

While a detailed structure activity relationship study is beyond thescope of this work, it is worth noting that the hydroxymethyl group isimportant for activity, since the absence of the OH group (as incompound 2) or the absence of any substituent at the five position ofthe furan (as in compound 3 results in a complete loss of activity.Orientation and opportunity for internal hydrogen-bonding appear to beimportant as well as can be seen from the weaker activity of 7 and 9compared to 1. At the same time, the lack of activity of 8 and the lowactivity of 4 make detailed analysis more difficult. Further studiesprobing the structural features responsible for activity and determininga molecular target will be reported in due course.

NF-κB is a major proinflammatory regulator that is frequently targetedfor anti-inflammatory drug discovery (Barnes and Karin 1997, Yamamotoand Gaynor 2001, Karin, Yamamoto et al. 2004). Although, the exactmechanism of aurone 1 suppression of NF-κB activity remains to beelucidated, we show that this novel compound can suppress thepro-inflammatory functions of both cultured human and murine macrophagecell lines without toxicity at effective doses. Therefore, we concludethat aurone 1 is an anti-inflammatory compound with therapeuticpotential for the possible treatment of chronic inflammatory disordersor conditions such as endotoxic shock that involves excessive TLR4/NF-κBsignaling.

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The detailed description and examples set forth herein have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artare also intended to be encompassed by the invention.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the invention. At the very least,and not as an attempt to limit the doctrine of equivalents to the scopeof the claims, each numerical parameter should at least be construed inlight of the number of reported significant digits and by applyingordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently, contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

Certain embodiments of the invention are mentioned below; however, thisdescription is not intended to be limiting in any way and is providedfor exemplary and illustrative purposes only. Other embodimentsencompassed by the invention are found throughout the disclosure.

In one aspect, the invention provides a method for treating orpreventing an infection, disease, or condition in a subject. The methodcan include administering to the subject a composition that includes aneffective amount of at least one substituted aurone.

In some embodiments, the infection, disease or condition is atrypanosomatid infection. The substituted aurone for treatment of atrypanosomatid infection can include a first component selected from thegroup consisting of a benzofuranone, an oxindole and a benzothiophenone,and a second component that includes an aryl group. In some embodiments,the substituted aurone can include at least one compound having FormulaI:

wherein Y is O, N or S;

Z is a substituted aryl group;

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and

a is 0, 1, 2, 3 or 4.

The trypanosomatid infection can include at least one of a Trypanosomabrucei infection, a Trypanosoma cruzi infection, or a Leishmaniainfection. The method can further include administering to the subjectan effective amount of at least one second compound. The second compoundcan include an antiprotozoan compound, an antiparasitic compound, or animmunomodulatory compound. Administration of the second compound occursbefore, after, or concurrent with administration of the substitutedaurone.

In some embodiments, the infection; disease or condition is a fungalinfection. The substituted aurone for treatment of a fungal infectioncan be(Z)-6,7-dihydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-one,having the structure

(Z)-2-(pyridin-2-ylmethylene)benzofuran-3(2H)-one, having the structure

or a combination thereof.

The fungal infection can include at least one of an infection withCandida spp., Cryptococcus spp., Saccharomyces cerevisiae orTrichophyton rubrum. The method can further include administering to thesubject an effective amount of at least one second compound. The secondcompound can include a systemic antifungal agent, a topical antifungalagent, or an immunomodulatory compound. Administration of the secondcompound can occur before, after, or concurrent with administration ofthe substituted aurone. The systemic antifungal agent or topicalantifungal agent can be an azole, a polyene, 5-fluorocytosine, or anechinocandin.

In some embodiments, the infection, disease or condition is infection,disease, or condition is an immune-related disease, disorder, orcondition, or other inflammatory condition. The substituted aurone fortreatment of an immune-related disease, disorder, or condition, or otherinflammatory condition can include at least one compound having FormulaII:

wherein R₁ is —CH₂OR₄, —CH₂NR₄R₅, —CH₂SR₄, —COR₄, or —CO₂R₄; R₂ and R₃are each independently selected from H, —CH₃, —CH₂OH, —OH or —OCH₃; R₄is H or alkyl; R₅ is H or alkyl; and X and Y are independently selectedfrom O, N and S.

The immune-related disease, disorder, or condition or other inflammatorycondition can be at least one of an autoimmune disease or inflammatorydisease. The autoimmune disease or inflammatory disease can berheumatoid arthritis or an inflammatory bowel disease. The method canfurther include administering to the subject an effective amount of atleast one second compound. The second compound can include animmunomodulatory compound, Administration of the second compound canoccur before, after, or concurrent with administration of thesubstituted aurone.

The composition administered to the subject can further include apharmaceutically acceptable carrier. The subject can be a human or ananimal, such as a companion animal, a domesticated animal, a wildanimal, or a zoo animal. For example, the animal can be a dog or a cow.

In another aspect, the invention provides a composition that includes atleast one substituted aurone and a pharmaceutically acceptable carrier.

In one embodiment of the composition, formulated for use in treating atrypanosomid infection, the aurone can include a first componentselected from the group consisting of a benzofuranone, an oxindole and abenzothiophenone, and, as a second component, an aryl group. Thesubstituted aurone used in the composition can include at least onecompound having Formula I:

wherein Y is O, N or S;

Z is a substituted aryl group;

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and

a is 0, 1, 2, 3 or 4.

The composition can include as a first active agent, the substitutedaurone, and a second active agent that includes at least one of anantiprotozoan compound or an antiparasitic compound.

In another embodiment of the composition, formulated for use in treatinga fungal infection, the substituted aurone can include(Z)-6,7-dihydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-onehaving the structure

(Z)-2-(pyridin-2-ylmethylene)benzofuran-3(2H)-one having the structure

or a combination thereof.

The composition can include, as first active agent, the substitutedaurone, and a second active agent that includes at least one a secondactive agent that includes at least one of an azole, a polyene,5-fluorocytosine, or an echinocandin,

In another embodiment of the composition, formulated for use in treatingan immune-related disease, disorder, or condition, or other inflammatorycondition, the substituted aurone can include at least one compoundhaving Formula II:

wherein R₁ is —CH₂OR₄, —CH₂NR₄R₅, —CH₂SR₄, —COR₄, or —CO₂R₄; R₂ and R₃are each independently selected from H, —CH₃, —CH₂OH, —OH or —OCH₃; R₄is H or alkyl; R₅ is H or alkyl; and X and Y are independently selectedfrom O, N and S.

The composition can be manufactured a controlled release formulation.

The composition may include, as a first active agent, the substitutedaurone, and may include a second active agent. The second active agentcan be at least one of an anti-inflammatory agent, a cytokine, achemokine, a therapeutic antibody, an immunogen, an antigen, anadjuvant, or an antioxidant, an immunomodulatory compound, an analgesic,a non-steroidal anti-inflammatory drug, a biologic compound, anantineoplastic agent, anticancer agent, antiangiogenic agent, achemopreventive agent, or a chemotherapeutic agent. the immunomodulatorycompound can be selected from the group consisting of azathioprine,6-mercaptopurine, cyclosporine A, tacrolimus, methotrexate,hydroxychloroquine, leflunomide, sulfasalazine, and minocycline.Additionally or alternatively, the immunomodulatory compound can be animmunomodulatory plant compound, such as curcumin, resveratrol,epigallocatechin, quercetin, capsaicin, colchicine, andrographolide,genistein, cis-gnetin H or trans-gnetin H.

The composition to be administered to the subject can includes least onenon-naturally occurring active agent.

Some embodiments of the methods, compositions, and compounds describedherein include the substituted aurone having Formula I:

wherein Y is O, N or S;

Z is a substituted aryl group;

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and

a is 0, 1, 2, 3 or 4.

In some embodiments of the substituted aurone of Formula I, Z isselected from the following substituted aryl groups:

wherein X is independently selected from C, O, N, and S;

R1 is selected from substituted or unsubstituted alkyl; substituted orunsubstituted aryl; substituted or unsubstituted alkoxy, hydroxyl,halogen; nitro; cyano, amine, ester, or combinations thereof; and

b is 0 1, 2, 3, or 4.

In some embodiments of the substituted aurone of Formula I, Z isselected from the following substituted aryl groups:

wherein R1 is selected from halogen, cyano, halogen substituted alkyl,or combinations thereof;

b is 1 or 2;

R2 is halogen; and

a is 0 or 1.

A substituted aurone having antitrypanosomal activity, useful in themethods and compositions of the invention, can be selected from thegroup consisting of compounds 6620, 6621, 4001, 2014, 9251, 9059, 9087,9019, 3002, 9024, 2023, 9030, 3005, 9028, 7000, 9062, 9065, 3004, 9084,9067, 2004, 2021, 9070, AA8, 2026, 9006, 9057, AA5A, TA2, AA4A, 3001,9312, AA3A, AA9, 2011, 9063, 3012, 6003, 9060, 9078, 9252, 9068, 9061,2015, 9056, AA11, 9086, 7002, 9053, 3009, 9076, 3011, 9058, 8002, 2013,9029, 6601, 3008, 4005, 6617, 2909, 4004, 9064, 9085, 5006, 2904, 9051,8001, 2911, 2018, 6001, 9253, 6000, 9050, 9088, 1009, 4006, 2001, 9007,2008, 2906, and 1001 as in Table 1. In some embodiments wherein thetrypanosomatid infection is a Trypanosoma brucei infection, thesubstituted aurone can be selected from the group consisting ofcompounds 6620, 6621, 4001 and 2014 as in Table 1 In some embodimentswherein the trypanosomatid infection is a Trypanosoma cruzi infection,the substituted aurone can be selected from the group consisting ofcompounds 9251, 9059, 9087, 9019, 3002, 9024, 2023, 9030, 3005, 9028,7000, 9062, 9065, 3004, and 9084 as in Table 1. In some embodimentswherein the trypanosomatid infection is Leishmania infection, andwherein the substituted aurone is selected from the group consisting ofcompounds 2023, 9030, 9067, 2004, 2021, 9070, AA8, 2026, 9006, 9057,AA5A, 6620, TA2, AA4A, 3001, 9312, AA3A, AA9, 2011, 9063, 3012, 6003,9060, 9065, 9078, 9252, 9068, 9087, 9062, 9061, 2015, 9056, AA11, 9086,7002, 9053, 9251, 3009, 9076, 9028, 3011, 9058, 8002, 9084, 2013, 9029,6601, 3008, 4005, 6617, 9059, 2909, 4004, 9064, 9085, 5006, 2904, 9051,8001, 3002, 2911, 2018, 6001, 9253, 6000, 9050, 9088, 1009, and 4006 asin Table 1. Particularly useful substituted aurones, which exhibitactivity against more than one pathogen, include compounds 2023, 3002,6620, 9028, 9030, 9059, 9062, 9065, 9084, 9087, and 9251 as in Table 1.

In another aspect, the invention provides a substituted aurone havingFormula if

wherein R₁ is —CH₂OR₄, —CH₂NR₄R₅, —CH₂SR₄, —COR₄, or —CO₂R₄; R₂ and R₃are each independently selected from H, —CH₃, —CH₂OH, —OH or —OCH₃; R₄is H or alkyl; R₅ is H or alkyl; and X and Y are independently selectedfrom O, N and S.

Some embodiments of the methods, compositions, and compounds describedherein include the substituted aurone having Formula II as shown above.Illustrative embodiments of the substituted aurone of Formula II areFormula II wherein:

(a) R₂=R₃=H; or

(b) R₁ is —CH₂OR₄; or

(c) R₄=H; or

(d) at least one of X and Y is O; or

(e) X=Y=O; or

(f) any combination of two, three, four or all of (a), (b), (c), (d),and

One such illustrative embodiment is(Z)-2-((5-(hydroxymethyl)furan-2-yl)methylene)benzofuran-3(2H)-one.

In another aspect, the invention provides a kit that includes, as anactive agent, a substituted aurone; and instructions for use. The activeagent is formulated for use in treating a trypanosomid infection, afungal infection, or an immune-related disease; disorder, or condition,or other inflammatory condition. The kit can include a pharmaceuticallyacceptable carrier. The kit can include at least one second active agentwhich can be co-administered with the substituted aurone.

In another aspect, the invention provides a substituted aurone, asdescribed throughout this disclosure, for use as an active agent fortreatment or prevention of a trypanosomatid infection, a fungalinfection, or an immune-related disease, disorder, or condition, orother inflammatory condition. The invention likewise provides for theuse of a substituted aurone for manufacture of a medicament for thetreatment or prevention of a trypanosomatid infection, a fungalinfection, or an immune-related disease, disorder, or condition, orother inflammatory condition.

In another aspect, the invention provides a substituted aurone thatincludes, as a first component, a benzofuranone; and as a secondcomponent, an aryl group. The substituted aurone has Formula I:

wherein Z selected from

where R1 is selected from iodine (I) or trifluoromethyl (CF₃), and b is1, 2 or 3;

where R1 is selected from alkyl, hydroxyl substituted alkyl, orcombinations thereof and b is 1 or 2;

where R1 is selected from halogen, or combinations thereof and b is 1 or2; and

R2 is substituted or unsubstituted alkyl, substituted or unsubstitutedalkoxy hydroxyl, halogen, or combinations thereof; and

a is 0, 1, 2, 3 or 4.

The first component can include one or more substituents; additionallyor alternatively, the second component can include one or moresubstituents.

In another aspect, the invention provides compound(Z)-6,7-dihydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-one,a substituted aurone having the structure

The invention also provides a method for making compound(Z)-6,7-dihydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-onethat includes mixing equimolar amounts of mixing equimolar amounts of6,7-dihydroxybenzofuranone and 3-hydroxy-4-methoxybenzaldehyde inmethanol; adding aqueous potassium hydroxide to the mixture; microwavingthe mixture at 110° C. for 12 minutes; washing the mixture with ethylacetate; neutralizing the mixture with acid to yield a crude solid; andwashing the crude solid with diethyl ether to yield the substitutedaurone.

In another aspect, the invention provides compound(Z)-2-(pyridin-2-ylmethylene)benzofuran-3(2H)-one, a substituted auronehaving the structure

The invention also provides a method for making(Z)-2-(pyridin-2-ylmethylene)benzofuran-3(2H)-one that includes mixingequimolar amounts of coumaranone and pyridine-2-carboxaldehyde in a dryvial; adding neutral alumina to the mixture; solvating the mixture withdichloromethane; reacting the mixture for hours at 25° C.; and filteringthe mixture to yield a crude solid; and purifying the crude solid usingethyl acetate/hexane eluent in column chromatography to yield thesubstituted aurone.

In another aspect, the invention provides compound(Z)-2-((5-(hydroxymethyl)furan-2-yl)methylene)benzofuran-3(2H)-one, asubstituted aurone having the structure

The invention also provides a method for making(Z)-2-(pyridin-2-ylmethylene)benzofuran-3(2H)-one that includes mixingequimolar amounts of coumaranone and 5-hydroxymethylfufural; adding a1:2 molar ratio of choline chloride:urea to the mixture; microwaving themixture at 90° C. for 30 minutes; partitioning the mixture between waterand methylene chloride; drying the organic layer; and purifying thedried organic layer by trituration with ether to yield the substitutedaurone.

The complete disclosures of all patents, patent applications includingprovisional patent applications, publications including patentpublications and nonpatent publications, and electronically availablematerial (including, for example, nucleotide and amino acid sequencesreported in databases such as GenBank, RefSeq, TPA, SwissProt, ProteinInformation Resource (KR) Protein Research Foundation (PRF), and ProteinData Bank (PDB)) which are cited herein are incorporated by reference.

1. A method for treating or preventing an infection, disease, or acondition in a subject, wherein the condition comprises a fungalinfection, the method comprising administering to the subject acomposition comprising an effective amount of at least one compoundhaving Formula I:

wherein: Y is O, N or S; Z is

wherein: X is C, O, N, or S; R1 is substituted or unsubstituted alkoxy,hydroxyl, halogen, or combinations thereof; and b is 1, 2, 3 or 4; R2 issubstituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and a is 0, 1, 2, 3 or
 4. 2-13. (canceled)
 14. The method of claim 1,wherein the composition further comprises a pharmaceutically acceptablecarrier.
 15. The method of claim 1, wherein the subject is a human. 16.The method of claim 1, wherein the subject is an animal. 17-18.(canceled)
 19. A composition comprising: at least one compound havingFormula I:

wherein: Y is O, N or S, Z is

wherein: X is C, O, N, or S; R1 is substituted or unsubstituted alkoxy,hydroxyl, halogen, or combinations thereof; and b is 1, 2, 3 or 4; R2 issubstituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and a is 0, 1, 2, 3 or 4; and a pharmaceutically acceptable carrier;wherein the composition is formulated for use in treating a fungalinfection. 20-31. (canceled)
 32. The method of claim 1, wherein: Z is

wherein: R1 is unsubstituted alkoxy, hydroxyl, or combinations thereof;and b is 1, 2, or 3; R2 is unsubstituted alkyl, hydroxyl, orcombinations thereof; and a is 0, 1, or
 2. 33. The method of claim 2,wherein the substituted aurone is selected from the group consisting ofcompounds 2011, 3012, 9078, 9252, 9068, 9056, 9053, 4005, 4004, 2911,9088, 1009, 2001, 6601, 6620, 2002, 4003, and 9055 as in Table
 1. 34-40.(canceled)
 41. A kit comprising an active agent comprising a compoundhaving Formula I:

wherein: Y is O, N or S; Z is

wherein: X is C, O, N, or S; R1 is substituted or unsubstituted alkoxy,hydroxyl, halogen, or combinations thereof; and b is 1, 2, 3 or 4; R2 issubstituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted alkoxy, hydroxyl,halogen, amine, cyano, nitro, azido, ethers, or combinations thereof;and a is 0, 1, 2, 3 or 4; and instructions for use; wherein the activeagent is formulated for use in treating a fungal infection. 42-54.(canceled)
 55. The method of claim 32, wherein the compound of Formula Iis(Z)-6,7-dihydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-one,having the structure


56. The method of claim 1, further comprising administering to thesubject an effective amount of at least one second compound comprisingan antiprotozoan compound, an antiparasitic compound, or animmunomodulatory compound, wherein administration of the second compoundoccurs before, after, or concurrent with administration of the compoundof Formula I.
 57. The method of claim 1, wherein the fungal infectioncomprises at least one of an infection with Candida spp., Cryptococcusspp., Saccharomyces spp. or Trichophyton spp.
 58. The method of claim57, further comprising administering to the subject an effective amountof at least one second compound comprising a systemic antifungal agent,a topical antifungal agent, or an immunomodulatory compound, whereinadministration of the second compound occurs before, after, orconcurrent with administration of the compound of Formula I.
 59. Thecomposition of claim 19, wherein: R2 is unsubstituted alkyl, hydroxyl,or combinations thereof; a is 0, 1, or 2; and Z is

wherein: R1 is unsubstituted alkoxy, hydroxyl, or combinations thereof;and b is 1, 2, or
 3. 60. The composition of claim 19 comprising a firstactive agent comprising the compound of Formula I, and furthercomprising a second active agent comprising at least one of an azole, apolyene, 5-fluorocytosine, or an echinocandin,
 61. The composition ofclaim 19 in a controlled release formulation.
 62. The composition ofclaim 19 further comprising a second active agent comprising at leastone of an anti-inflammatory agent, a cytokine, a chemokine, atherapeutic antibody, an immunogen, an antigen, an adjuvant, or anantioxidant, an immunomodulatory compound, an analgesic, a non-steroidalanti-inflammatory drug, a biologic compound, an antineoplastic agent,anticancer agent, antiangiogenic agent, a chemopreventive agent, or achemotherapeutic agent.
 63. The composition of claim 62, wherein theimmunomodulatory compound is selected from the group consisting ofazathioprine, 6-mercaptopurine, cyclosporine A, tacrolimus,methotrexate, hydroxychloroquine, leflunomide, sulfasalazine, andminocycline.
 64. The kit of claim 41 further comprising at least onesecond active agent which can be co-administered with the compound ofFormula I.
 65. The composition of claim 59, wherein the compound ofFormula I is(Z)-6,7-dihydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-one,having the structure


66. The kit of claim 41, wherein: R2 is unsubstituted alkyl, hydroxyl,or combinations thereof; a is 0, 1, or 2; and Z is

wherein: R1 is unsubstituted alkoxy, hydroxyl, or combinations thereof;and b is 1, 2, or
 3. 67. The kit of claim 66, wherein the compound ofFormula I is(Z)-6,7-dihydroxy-2-(3-hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-one,having the structure